Compositions and methods for phagocyte delivery of anti-staphylococcal agents

ABSTRACT

The present application generally relates to multi-specific molecules that bind to multiple bacterial virulence factors, methods for producing these binding molecules, and the use of these binding molecules to treat bacterial infections. In particular, the binding molecules comprise at least two binding domains, preferably an antibody or antibody fragment and an alternative scaffold. The first binding domain is capable of binding to a glycosylated staphylococcal surface protein, preferably an SDR-containing protein. The second binding domain is capable of binding to a staphylococcal leukotoxin, preferably LukAB, LukD or LukE. These multi-specific binding compounds have killing activity against staphylococci and, thus, can be used in the treatment and/or amelioration of a  Staphylococcus  infection, including methicillin-resistant  Staphylococcus aureus.

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/913,714, filed Dec. 9, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to multi-specific binding molecules that target at least two different bacterial virulence factors. In particular, the binding molecules comprise at least two binding domains that specifically bind to different staphylococcal proteins (i.e., the molecules are at least bispecific). The first binding domain, preferably an antibody or a fragment thereof, is capable of binding to a glycosylated staphylococcal surface protein, preferably glycosylated serine-aspartate dipeptide repeat (SDR) containing proteins. The second binding domain, preferably an alternative scaffold, is capable of binding to a staphylococcal leukotoxin, preferably LukAB, LukD or LukE. These multi-specific binding compounds have killing activity against staphylococci and, thus, can be used in the prevention, treatment and/or amelioration of a Staphylococcus aureus infection, including methicillin-resistant Staphylococcus aureus (MRSA) infections.

BACKGROUND

Bacterial infections caused by staphylococcus bacteria (i.e., a “staph infection”) are very common in the general population. About 25% of individuals commonly carry staphylococcus bacteria on their skin or in their nose. Most of the time, these bacteria cause no problem or result in relatively minor skin infections. However, staph infections can turn deadly if the bacteria invade deeper into an individual's body, for example, entering the bloodstream, joints, bones, lungs or heart. In the past, a lethal staph infection might have occurred in a person who was hospitalized or had a chronic illness or weakened immune system. Now, it is increasingly common for an otherwise healthy individual to develop life-threatening staph infections. Importantly, many staph infections no longer respond to common antibiotics.

Staphylococcus aureus, often referred to as “staph,” Staph. aureus,” or “S. aureus,” is a major human pathogen, producing a multitude of virulence factors making it able to cause several types of infection, from superficial lesions to toxinoses and life-threatening systemic conditions such as endocarditis, osteomyelitis, pneumonia, meningitis and sepsis (reviewed in Miller and Cho, “Immunity Against Staphylococcus aureus Cutaneous Infections,” Nat. Rev. Immunol. 11:505-518 (2011)). Although most individuals encounter S. aureus shortly after birth (Holtfreter et al., “Towards the Immune Proteome of Staphylococcus aureus—The Anti-S. aureus Antibody Response,” Int. J. Med. Microbiol. 300:176-192 (2010)) and possess both antibodies against S. aureus and the ability to increase anti-S. aureus titers after infection, these antibodies are often not protective against recurrent S. aureus infections (Foster T J, “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005)). In the United States alone, an annual mortality of more than 20,000 is attributed to methicillin-resistant S. aureus (MRSA), exceeding deaths caused by influenza, viral hepatitis, and HIV/AIDS (Foster, T J., “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005); Klevens et al., “The Impact of Antimicrobial-Resistant, Health Care-Associated Infections on Mortality in the United States,” Clin. Infect. Dis. 47:927-930 (2008)). The pathogen produces a variety of molecules that presumably facilitate survival in or on the human host.

Bi-component, pore-forming leukotoxins are among the secreted virulence factors produced by S. aureus. These toxins can be secreted as water soluble monomers which oligomerize, and then insert pores into the plasma membrane, which subsequently disrupt the cellular osmotic balance and membrane potential leading to death of the targeted cells, most notably polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes (Bischogberger et al., “Pathogenic Pore-Forming Proteins: Function and Host Response,” Cell Host Microbe 12(3):266-275 (2012), which is hereby incorporated by reference in its entirety). In the case of Leukotoxin ED (LukED), the targeting, binding, and killing of host phagocytic cells occurs via the cellular target CCR5, CXCR1 and CXCR2 located on the surface of the phagocytes (Alonzo III et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83:423-435 (2012); Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430)51-55 (2012); and Reyes-Robles et al., “Staphylococcus aureus Leukotoxin ED Targets the Chemokine Receptors CXCR1 and CXCR2 to Kill Leukocytes and Promote Infection,” Cell Host & Microbe 14:453-459 (2013)). Indeed, when the cellular target of LukED, CCR5, is not present on host immune cells, the host animal is resistant to the otherwise lethal S. aureus infection (Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2012)). Leukotoxin AB (LukAB) can also kill host phagocytic cells, and its cytolytic activity can be exerted both from the outside and from the inside of the cell, after the microorganism is phagocytosed into the host cell (Dumont et al., “Staphylococcus aureus LukAB Cytotoxin Kills Human Neutrophils by Targeting the CD11b Subunit of the Integrin Mac-1,” PNAS 110(26):10794-10799 (2013)). Due to the contribution that both of these leukotoxins have to pathogenesis, they have been considered critical S. aureus virulence factors (Alonzo III and Tones, “Bacterial Survival Amidst an Immune Onslaught: The Contribution of the Staphylococcus aureus Leukotoxins,” PLOS Path 9(2):e1003143 (2013)).

Another critical factor for the pathogenic success of S. aureus depends on the properties of its surface proteins (Clarke et al., “Surface Adhesins of Staphylococcus aureus,” Adv. Microb. Physiol. 51:187-224 (2006); Patti et al., “MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,” Annu. Rev. Microbiol. 48:585-617 (1994); and Patti et al., “Microbial Adhesins Recognizing Extracellular Matrix Macromolecules,” Curr. Opin. Cell Biol. 6:752-758 (1994)).

S. aureus employs microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in order to adhere to and colonize host tissues. MSCRAMMs can recognize collagen, heparin-related polysaccharides, fibrinogen, and/or fibronectin. S. aureus expresses a subset of MSCRAMMs, which all contain the serine-aspartate dipeptide repeat (SDR) domain, including clumping factor A (ClfA), clumping factor B (ClfB), SdrC, SdrD, and SdrE (Becherelli et al. “Protective Activity of the CnaBE3 Domain Conserved Among Staphylococcus aureus Sdr Proteins,” PLoS One 8(9): e74718 (2013)). S. epidermidis also expresses three members of this family, SdrF, SdrG, and SdrH (McCrea et al., “The Serine-Aspartate Repeat (Sdr) Protein Family in Staphylococcus Epidermidis,” Microbiology 146:1535-1546 (2000)). All of these proteins share a similar structure comprising an N-terminal ligand-binding A domain followed by the SDR domain, which can contain between 25-275 serine-aspartate dipeptide repeats. The C-terminal portion of these proteins contains the LPXTG-motif, which facilitates cell wall anchoring by the transpeptidase sortase A. The serine-aspartate dipeptide regions in SDR-containing proteins are modified by the sequential addition of glycans by two glycosyltransferases. First, SdgB appends N-acetylglucosamine (GlcNAc) on serine residues within the serine-aspartate dipeptide regions, followed by SdgA modification of the glycoprotein, resulting in disaccharide moieties. This glycosylation can protect SDR-containing staphylococcal proteins from Cathepsin G-mediated degradation (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog 9(10):e1003653 (2013)).

Additionally, Protein A, also located on the surface of S. aureus, contributes to staphylococcal escape from protective immune responses by capturing the Fc domain of host IgG, as well as the Fab domain of the VH3 clan of IgG and IgM (Sjodahl et al., “Repetitive Sequences in Protein A from Staphylococcus aureus. Arrangement of Five Regions Within the Protein, Four Being Highly Homologous and Fc-Binding,” Eur. J. Biochem. 73:343-351 (1997); and Cary et al., “The Murine Clan V(H) III Related 7183, J606 and S107 and DNA4 Families Commonly Encode for Binding to a Bacterial B cell Superantigen,” Mol. Immunol. 36:769-776 (1999)).

Additionally, S. aureus expresses a second immunoglobulin binding protein referred to as the second binding protein for immunoglobulins (Sbi) (Zhang et al., “A Second IgG-Binding Protein in Staphylococcus aureus,” Microbiology 144:985-991 (1998) and Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)) that is either secreted or associated with the cell envelope (Smith et al., “The Sbi Protein is a Multifunctional Immune Evasion Factor of Staphylococcus aureus” Infection & Immunity 79:3801-3809 (2011) and Smith et al., “The Immune Evasion Protein Sbi of Staphylococcus aureus Occurs both Extracellularly and Anchored to the Cell Envelope by Binding to Lipotechoic Acid” Mol. Microbiol. 83:789-804 (2012)) and shares a pair of conserved helices with Protein A involved in binding to the Fc region of IgG proteins (Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)).

Furthermore, S. aureus secretes a number of proteases that have been implicated in immune evasion. Rooijakkers et al. demonstrated that S. aureus secretion of staphylokinase, a plasminogen activator protein, led to the activation of plasmin that cleaved both surface-bound IgG and complement C3b, ultimately reducing immune-mediated S. aureus destruction (Rooijakkers et al., “Anti-Opsonic Properties of Staphylokinase,” Microbes and Infection 7:476-484 (2005)). S. aureus also secretes the serine protease glutamyl endopeptidase V8 (GluV8) that can directly cleave human IgG1 in the lower hinge region between E233 and L234 (EU numbering (Edelman et al., “The Covalent Structure of an Entire GammaG Immunoglobulin Molecule,” PNAS 63:78-85 (1969), Brerski et al., “Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs,” J. Immunol. 181:3183-3192 (2008)). It was also recently demonstrated that human anti-S. aureus IgGs are rapidly cleaved when bound to the surface of S. aureus (Fernandez Falcon et al., “Protease Inhibitors Decrease IgG Shedding From Staphylococcus aureus, Increasing Complement Activation and Phagocytosis Efficiency,” J. Med. Microbiol. 60:1415-1422 (2011)).

Taken together, these studies indicate that S. aureus utilizes a number of mechanisms that could adversely affect standard IgG1-based monoclonal antibody (mAb) therapeutics, either by directly cleaving the mAb, sequestering the mAb by Protein A binding, or by killing off the very effector cells required for therapeutic efficacy. It is therefore not surprising that presently there are no mAb-based therapies targeting S. aureus that have achieved final approval for use in humans. Thus, there remains a need for methods and compositions that can treat staphylococcal infection, which (i) evade protein A and Sbi binding, (ii) escape staph-induced proteolysis, (iii) can neutralize leukotoxins and (iv) are capable of opsonizing and delivering S. aureus to phagocytes. The present application meets these and other needs.

Summary

The present disclosure provides multi-specific binding molecules comprising at least a first binding domain and a second binding domain, each of which specifically binds to a different bacterial virulence factor in particular a different staphylococcal virulence factor. The first and the second binding domains are optionally connected via a linker peptide.

The first binding domain is capable of binding to a glycosylated staphylococcal surface protein, such as an staphylococcal SDR-containing protein. In one aspect, the staphylococcal SDR-containing protein is ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG and SdrH. Preferably, the staphylococcal SDR-containing protein is ClfA, ClfB, SdrC, SdrD or SdrE.

In one aspect, the first binding domain is a full-length antibody or antibody fragment. Preferably, the full-length antibody or antibody fragment is resistant to proteolytic degradation by a staphylococcal protease that cleaves wild-type IgG1 (such as a staphylococcal protease, e.g., Staphylococcus aureus V8 protease, that cleaves wild-type IgG1 between or at residues 222-237 (EU numbering) within SEQ ID NO:60). In another aspect, the full-length antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

In one aspect, the binding molecule is not capable of specific binding to human FcγRI, is not capable of specific binding to Protein A, and is not capable of specific binding to Sbi. In one aspect, the binding molecule is capable of specific binding to FcRn.

In one aspect, the first binding domain comprises an immunoglobulin heavy chain variable (VH) region having the amino acid sequence selected from the group of VH region amino acid sequences of SEQ ID NOs:60, 62, 64 or 66. In another aspect, the first binding domain comprises an immunoglobulin light chain variable (VL) region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. Alternatively, the first binding domain comprises (a) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (b) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. In one embodiment, the first binding domain comprises (1) a VH region having the amino acid sequence of SEQ ID NO:60, and a VL region having the amino acid sequence of SEQ ID NO:61; (2) a VH region having the amino acid sequence of SEQ ID NO:62, and a VL region having the amino acid sequence of SEQ ID NO:63; (3) a VH region having the amino acid sequence of SEQ ID NO:64, and a VL region having the amino acid sequence of SEQ ID NO:65; (4) a VH region having the amino acid sequence of SEQ ID NO:66, and a VL region having the amino acid sequence of SEQ ID NO:67; (5) a VH region having the amino acid sequence of SEQ ID NO:68, and a VL region having the amino acid sequence of SEQ ID NO:69; (6) a VH region having the amino acid sequence of SEQ ID NO:70, and a VL region having the amino acid sequence of SEQ ID NO:71; (7) a VH region having the amino acid sequence of SEQ ID NO:72, and a VL region having the amino acid sequence of SEQ ID NO:73; (8) a VH region having the amino acid sequence of SEQ ID NO:74, and a VL region having the amino acid sequence of SEQ ID NO:75; (9) a VH region having the amino acid sequence of SEQ ID NO:76, and a VL region having the amino acid sequence of SEQ ID NO:77; or (10) a VH region having the amino acid sequence of SEQ ID NO:78, and a VL region having the amino acid sequence of SEQ ID NO:79.

In yet another aspect, the binding molecule comprises (a) a first binding domain that comprises (i) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (ii) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67; and (b) a second binding domain that comprises the amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666. In once aspect, the second binding domain comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:22.

The second binding domain is capable of binding to a staphylococcal leukotoxin. In one aspect, the staphylococcal leukotoxin is selected from a group consisting of leukotoxin A (LukA), leukotoxin B (LukB), leukotoxin AB (LukAB), leukotoxin D (LukD), leukotoxin E (LukE), leukotoxin ED (LukED), Panton-Valentine leukocidin S (LukS-PV), Panton-Valentine leukocidin F (LukF-PV), Panton-Valentine leukocidin (LukSF/PVL), gamma hemolysin A (HlgA), gamma hemolysin C (HlgC), gamma hemolysin B (HlgB), gamma hemolysin AB (HlgAB), and gamma-hemolysin BC (HlgBC). In one aspect, the staphylococcal leukotoxin is LukAB, LukD, or LukE.

In one aspect, the second binding domain is an alternative scaffold. In one aspect, the alternative scaffold comprises a fibronectin type III (FN3) domain. In one aspect, the FN3 domain binds to LukA having the amino acid sequence of SEQ ID NO:10, LukB having the amino acid sequence of SEQ ID NO:11, LukD having the amino acid sequence of SEQ ID NO:12, or LukE having the amino acid sequence of SEQ ID NO:13. Alternatively, the FN3 domain binds to a LukAB complex comprising LukA having the amino acid sequence of SEQ ID NO:10 and LukB having the amino acid sequence of SEQ ID NO:11, and/or a LukED complex comprising LukE having the amino acid sequence of SEQ ID NO:13 and LukD having the amino acid sequence of SEQ ID NO:12.

In one aspect, the second binding domain of the binding molecule competes with an FN3 domain having an amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666 for binding to (i) LukA having the amino acid sequence of SEQ ID NO:10; (ii) LukB having the amino acid sequence of SEQ ID NO:11; (iii) LukD having the amino acid sequence of SEQ ID NO:12; and/or (iv) LukE having the amino acid sequence of SEQ ID NO:13.

In one aspect, the second binding domain is a FN3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:14-59 and SEQ ID NOs:113-666. In one aspect, the second binding domain is a FN3 domain comprising the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:22.

In one aspect, the binding molecule further comprises one or more additional binding domains, where the one or more additional binding domains are capable of (i) binding to a different glycosylated staphylococcal surface protein than bound by the first binding domain and/or (ii) binding to a different staphylococcal leukotoxin than bound by the second binding domain.

The disclosure also provides nucleic acid sequences encoding the above described binding molecules as well as vectors comprising a nucleic acid sequence or nucleic acid sequences. Additionally, the invention contemplates a host cell comprising these vectors.

Moreover, the disclosure provides a process for the production of the above described binding molecules, comprising (a) culturing a host cell containing a vector comprising a nucleic acid sequence or nucleic acid sequences that encode the inventive binding molecules, and (b) recovering the binding molecule from the culture.

Furthermore, the disclosure provides a pharmaceutical composition comprising a binding molecule as described herein.

The binding molecules provided herein can be used in the treatment, prevention or amelioration of a staphylococcal infection. In one aspect, the staphylococcal infection is caused by Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Accordingly, the present disclosure provides a method for the treatment, prevention or amelioration of a staphylococcal infection, comprising (a) administering to a subject in need thereof a binding molecule as described herein. This method of treatment, prevention of amelioration of staphylococcal infection may involve repeated administration of the binding molecule as described herein, or administration of the binding molecule in combination with one or more other agents, such as an anti-infective agent, an antibiotic agent, and/or an antimicrobial agent.

The binding molecules provided herein can also be used in methods for diagnosing a staphylococcal infection in a subject. In one aspect, the method for diagnosing a staphylococcal infection involves contacting the binding molecule as described herein with a sample from a subject and detecting the presence or the absence of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample based on the contacting. This method further involves diagnosing the staphylococcal infection in the subject based on the detection of the glycosylated staphylococcal surface protein and/or the staphylococcal leukotoxin in the sample.

Similar methods can be employed for the detection of a staphylococcus in a sample. In particular, the methods for detection of staphylococcus in a sample involve contacting the binding molecule as described herein with a sample, and detecting the presence or the absence of a glycosylated staphylococcus surface protein and/or a staphylococcal leukotoxin in the sample based on the contacting, whereby the presence of the glycosylated staphylococcus surface protein and/or staphylococcal leukotoxin indicates the presence of staphylococcus in the sample.

Furthermore, the binding molecules of the present invention can be used for the prevention of a staphylococcal infection. In one aspect, a method for the prevention of a staphylococcal infection involves contacting the binding molecule described herein with a sample from the subject, and detecting a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample as a result of the contacting. The method further involves administering an agent suitable for preventing staphylococcal infection to the subject based on the detection of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample.

Also contemplated by the present disclosure is a kit comprising a binding molecule as provided herein, and a kit comprising a nucleic acid molecule encoding the binding molecules provided, a vector comprising a nucleic acid molecule(s) encoding the binding molecules provided and/or a host cell containing such a vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the characterization of anti-LukD FN3 domains. LukED (75 nM) was mixed with increasing concentrations of the indicated FN3 domains for 30 minutes prior mixing with hPMNs. Cells were intoxicated for 1 hr. Data was generated from at least three independent blood donors, and error bars represent mean±SEM. FIG. 1A shows data obtained from Cell Titer measurement (cell metabolism). FIG. 1B shows obtained from LDH measurements (membrane damage).

FIGS. 2A and 2B show the characterization of anti-LukAB FN3 domains. LukAB (18.8 nM) was mixed with increasing concentrations of the indicated FN3 domains for 30 minutes prior mixing with hPMNs. Cells were intoxicated for 1 hr. Data was generated from at least three independent blood donors, and error bars represent mean±SEM. FIG. 2A shows data obtained from ethidium bromide permeability assay (EtBr) (pore-formation/membrane permeability). FIG. 2B shows data obtained from LDH measurements (membrane damage).

FIG. 3 shows the characterization of anti-LukE FN3 domains. LukED (75 nM) was mixed with increasing concentrations of the indicated FN3 domains for 30 minutes prior mixing with hPMNs. Cells were intoxicated for 1 hr. Data obtained from LDH measurements (membrane damage). Data was generated from at least three independent blood donors, and error bars represent mean±SEM.

FIGS. 4A-4F depict exemplary multispecific binding proteins containing an antibody variable region that targets a Staph antigen conjugated to a leukotoxin neutralizing domain. FIG. 4A shows an exemplary multispecific binding protein containing a monospecific, bivalent anti-Staph mAb variable domain and a heavy chain C-terminal conjugated LukAB, LukD or LukE neutralizing domain. FIG. 4B illustrates an exemplary multispecific binding protein containing a monospecific, bivalent anti-Staph mAb variable domain and a light chain C-terminal conjugated LukAB, LukD or LukE neutralizing domain. FIG. 4C shows an exemplary multispecific binding protein containing a monospecific, bivalent anti-Staph mAb variable domain as well as a light chain C-terminal conjugated LukAB, LukD or LukE neutralizing domain and a heavy chain C-terminal conjugated LukAB, LukD or LukE neutralizing domain. FIG. 4D shows an exemplary multispecific binding protein containing a monospecific, bivalent anti-Staph mAb variable domain and a heavy chain C-terminal conjugated LukAB, LukD or LukE neutralizing domain with a tandem C-terminal conjugated LukAB, LukD or LukE neutralizing domain. FIG. 4E show an exemplary multispecific binding protein containing a bispecific anti-Staph mAb variable domain and a heavy chain C-terminal conjugated LukAB neutralizing domain and a heavy chain C-terminal conjugated LukAB neutralizing domain and a heavy chain C-terminal conjugated LukD or LukE neutralizing domain. FIG. 4F illustrates an exemplary multispecific binding protein containing a monospecific, bivalent anti-Staph mAb and a heavy chain C-terminal conjugated LukAB neutralizing domain and a heavy chain C-terminal conjugated LukD or LukE neutralizing domains.

FIG. 5 shows that mAb 5133 recognizes a SdgB-glycosylated SDR protein of S. aureus. mAb 5133 does not detect high molecular weight bands in a sdgB mutant strain of S. aureus as assessed by western blotting of lysates from different S. aureus strains.

FIG. 6 shows that mutation of the lower hinge region of IgG1 conferred increased resistance to GluV8 digestion. PRASA constructs with a mutated lower hinge region, without or with a FN3 domain on the C terminus of the heavy chain (construct 4 and construct 6, respectively), are resistant to proteolysis by the S. aureus protease GluV8 compared to constructs containing the wildtype (wt) human IgG1 lower hinge region (IgG1 isotype control and construct 3).

FIGS. 7A-7D show that the addition of FN3 domains does not affect PRASA variant binding to human FcγRs. Competition ALPHASCREEN® assays were employed in order to assess the ability of anti-Staph targeting constructs to bind to FcγRs. As shown in the graph of FIG. 7A, PRASA constructs (constructs 4-7) do not show detectable binding to FcγRI. All constructs (IgG1 isotype control and constructs 3-7) show similar binding to human FcγRIIa (R131) as shown in FIG. 7B. The graph of FIG. 7C shows PRASA constructs (constructs 4-7) have decreased binding to human FcγRIIb compared to IgG1 and A6 constructs (IgG1 isotype control and construct 3, respectively). As shown in the graph of FIG. 7D, all constructs (IgG1 isotype control and constructs 3-7) show similar binding to human FcγRIIIa (V158).

FIG. 8 shows that the CH3 mutations H435R/Y436F reduced and/or eliminated Protein A binding, as determined using direct Protein A ELISA assays. PRASA A6 variants (constructs 4-7) do not show detectable binding to Protein A in a plate-based ELISA assay, whereas human IgG1 (IgG1 isotype control) binds to Protein A.

FIG. 9 shows that mAb-FN3 domain binding conjugates are capable of binding to S. aureus (Newman strain) in a flow cytometry-based binding assay.

FIGS. 10A-10B show the characterization of the mAb-FN3 domain conjugates. LDH measurements (membrane damage) were taken from at least three independent blood donors, and error bars represent mean±SEM. The graph of FIG. 10A shows LDH release from hPMNs after exposure to LukED (75 nM) that was mixed with increasing concentrations of the indicated mAbs or FN3 domain-mAb conjugates for 30 minutes prior to cell exposure. Cells were intoxicated for 1 hr. The graph of FIG. 10B shows LDH release from hPMNs after exposure to LukAB (18.8 nM) that was mixed with increasing concentrations of the indicated mAbs or mAb-FN3 domain conjugates for 30 minutes prior mixing with hPMNs. Cells were intoxicated for 1 hr.

FIG. 11 shows that Staph LukAB-mediated cytotoxicity post-phagocytosis can be neutralized by the mAb-FN3 domain conjugates. Primary human PMNs were infected with Staph previously opsonized with different mAb 5133 antibodies or without antibodies. Opsonized bacteria were used to infect hPMNs at an MOI of 10 in the presence of ethidium bromide. The ability of Staph to induce membrane damage was evaluated by measuring ethidium bromide permeability over 2 hrs. Data was generated from at least three independent blood donors, and error bars represent mean±SEM.

FIG. 12 shows that mAb-FN3 domain fusion proteins bearing anti-LukAB FN3 domains neutralize leukotoxin LukAB and promote human polymorphonuclear neutrophil (PMN) mediated restriction of the growth of S. aureus. These experiments employed a variant of S. aureus strain Newman that harbors a plasmid that expresses the green fluorescent protein (gfp). Freshly grown GFP-labeled bacteria were normalized to 1×10⁷ CFU/mL and 1 mL aliquots pelleted by centrifugation and re-suspended in 300 μl of different mAb or mAb-FN3 fusion proteins at 1.5 mg/mL, or PBS (non-opsonized). Samples were brought up to 1 mL with RPMI-HH and then incubated for 20 minutes at 37° C. for opsonization. The opsonized bacteria were subsequently mixed with 2×10⁵ freshly isolated primary hPMNs at a multiplicity of infection (MOI) of ˜10:1 (bacteria:hPMN). Bacteria and cells were synchronized by centrifugation at 1500 RPM for 7 minutes and placed in a 37° C. 5% CO2 incubator. GFP fluorescence, as an indicator of bacterial growth was measured 5 hours post-infection. As shown in FIG. 12, these experiments demonstrate that in comparison to the IgG1 isotype control, both A6 construct 3 and PRASA A6 construct 4 promote hPMN-mediated S. aureus growth-restriction. This is consistent with the opsonic capability of the 5133 mAb. Importantly, the LukAB FN3 domain-mAb 5133 conjugates (PRASA A6 HC-AB construct 6 and PRASA A6 LC-D HC-AB construct 7) enhanced PMN-mediated growth restriction compared to the PRASA A6 parental mAb (construct 4). The lack of enhancement in growth restriction by the CR5133/anti-LukD FN3 domain-mAbs (PRASA A6 LC-D construct 5) is consistent with previous findings that LukED production is minimal during ex vivo infection models.

FIG. 13 shows SdgB-mediated glycosylation of a recombinant protein (SdrC4) bearing a Serine Aspartate Repeat (SDR) element results in creation of a specific epitope recognized by mAb 5133. Incubation of resin-bound SdrC4 protein with cell lysates prepared from S. aureus strains JE2 (FIG. 13, panel B, lanes 2 and 6) and an otherwise-isogenic sdgA mutant derivative (FIG. 13, panel B, lanes 3 and 7) results in the detection of a specific ˜95 kDa species by western blot employing mAb 5133. In contrast no such protein band was detected by mAb 5133 following incubation of resin-bound SdrC4 protein with lysates prepared from an sdgB mutant derivative of S. aureus strain JE2 that does not express the SdgB glycosyltransferase (FIG. 13, panel B, lanes 4 and 8), or in the control PBS sample (FIG. 13, panel B, lanes 1 and 5) FIG. 13, panel A shows a colloidal blue stain of the gel corresponding to the blot of FIG. 13B, indicating that essentially equivalent amounts of the recombinant SdrC4 protein were loaded in lanes 1-4 and 5-8.

FIGS. 14A-14C are a series of western blots showing that in vitro glycosylation of recombinant proteins bearing Serine Aspartate Repeat (SDR) elements with purified, recombinant SdgB glycosyltransferase protein results in the generation of an epitope recognized by mAb 5133. Recombinant SDR proteins (SdrC4 and SdrC5) bearing poly-histidine (His)₆ affinity tags were incubated with buffer alone, buffer plus uridine diphosphate N-acetylglucosamine (UDP-GlcNac), or buffer plus UDP-GlcNac plus a recombinant form of the S. aureus SdgB glycosyltransferase. Reaction products were separated by SDS-PAGE, transferred to PVDF membranes and the resulting western blots probed with either (i) mAb 5133 as the primary antibody and detection using a secondary HRP-conjugated goat anti-human IgG (H+L) detection antibody (panel A of FIGS. 14A, 14B and 14C), or (ii) a HRP-conjugated mAb specific for the poly-histidine (His)₆ affinity tag (panel B of FIGS. 14A, 14B, and 14C).

FIG. 15 shows that mAb5133 binds SdgB-glycosylated SdrC4 protein (FIG. 15, panel A), but does not bind purified S. aureus LTA (FIG. 15, panel B). In contrast, pagibaximab, a well characterized anti-LTA mAb, binds purified S. aureus LTA (panel B) but does not bind SdgB-glycosylated SdrC4 protein (panel A). ELISA format assays employed plate-immobilized, recombinant SdgB-glycosylated SdrC4 protein or purified S. aureus LTA and primary antibodies mAb 5133, pagibaximab or a mAb specific for the respiratory syncytial virus F (RSV-F) as a negative control for binding of either antigen.

FIG. 16 shows that mAb 5133 and variants bearing mutations that eliminate GluV8-mediated protease cleavage in the hinge region (PRASA) and/or protein-A binding (A6) retain binding to both the human (FIG. 16, panel A) and mouse (FIG. 16, panel B) FcRn receptors. ELISA format competition assays employed poly-histidine tagged recombinant forms of human or mouse FcRn receptor bound on copper-coated plates, and a human IgG1 antibody targeting the respiratory syncytial virus F (RSV-F) glycoprotein (CNTO 3929) as the competitor mAb.

FIG. 17 shows that mAb-FN3 domain fusion proteins bearing mutations that eliminate GluV8-mediated protease cleavage in the hinge region (PRASA) and/or protein-A binding (A6) retain binding to both the human FcRn receptor. ELISA format competition assays employed poly-histidine tagged recombinant form of the human FcRn receptor bound on copper-coated plates and a human IgG1 antibody targeting the respiratory syncytial virus F (RSV-F) glycoprotein (CNTO 3929) as the competitor mAb.

FIGS. 18A-18B show that variants of mAb 1533 bearing mutations that eliminate protein-A binding (A6) are unable to engage with either of the S. aureus IgG binding proteins, Protein-A or Sbi. Cell lysates were prepared from S. aureus strains JE2 (wild-type for expression of both Protein A and Sbi) and derivatives NE286 (a protein-A deficient spa mutant of JE2), NE453 (an sbi mutant of JE2 deficient in expression of the Sbi protein) and Wood 46 (a strain known to lack expression of Protein A), separated by SDS-PAGE, and transferred to PVDF membranes. The resulting Western blots were probed with either (i) mAb 5133 (FIG. 18, panel A), (ii) a mAb 1533 variant bearing A6 mutations that eliminate protein-A binding (FIG. 18A, panel B), or (iii) a mAb 1533 variant bearing both the A6 mutations plus mutations that eliminate GluV8-mediated protease cleavage in the hinge region (PRASA) (FIG. 18B, panel C) with secondary detection via a secondary antibody (goat anti-human IgG (H+L).

FIG. 19 shows the differential binding and neutralization of γ-hemolysin HlgAB by representative FN3 domain proteins Luk151 and Luk129. Binding of the FN3 domains to purified, recombinant HlgA was determined by ELISA. For leukotoxin neutralization studies, the FN3 domain test articles were incubated with purified, recombinant HlgAB, then freshly isolated primary human neutrophils were added and incubated for one hour at 37° C. The viability of the remaining neutrophils was determined by measuring the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. As is shown in FIG. 19, the FN3 domain variant Luk151 exhibits binding to the HlgA subunit but does not neutralize the cytolytic activity of the HlgAB leukotoxin. In contrast, Luk129 exhibits binding to the HlgA subunit and neutralizes the cytolytic activity of the HlgAB leukotoxin as exemplified by complete inhibition of LDH release.

FIG. 20 shows the differential binding and neutralization of γ-hemolysin HlgCB by representative FN3 domain proteins Luk357 and Luk188. Binding of the FN3 domains to purified, recombinant HlgC was determined by ELISA. For leukotoxin neutralization studies, the FN3 domain test articles were incubated with purified, recombinant HlgCB, then freshly isolated primary human neutrophils were added and incubated for one hour at 37° C. The viability of the remaining neutrophils was determined by measuring the release of LDH from cells with a damaged membrane. As is shown in FIG. 20, the FN3 domain variant Luk357 exhibits binding to the HlgC subunit but does not neutralize the cytolytic activity of the HlgCB leukotoxin. In contrast, Luk188 exhibits binding to the HlgC subunit and neutralizes the cytolytic activity of the HlgCB leukotoxin as exemplified by complete inhibition of LDH release.

FIG. 21 shows the differential binding and neutralization of the Panton-Valentine leukocidin (PVL) by representative FN3 domain proteins Luk540 and Luk647. Binding of the FN3 domains to purified, recombinant LukS-PV was determined by ELISA. For leukocidin neutralization studies, the FN3 domain test articles were incubated with purified, recombinant PVL leukocidin, then freshly isolated primary human neutrophils were added and incubated for one hour at 37° C. The viability of the remaining neutrophils was determined by measuring the release of LDH from cells with a damaged membrane. As shown in FIG. 21, the FN3 domain variant Luk540 exhibits binding to the LukS-PV subunit of the PVL leukotoxin but does not neutralize the cytolytic activity of PVL. In contrast, Luk647 exhibits binding to the LukS-PV subunit of the PVL leukotoxin and neutralizes the cytolytic activity of the PVL leukotoxin as exemplified by complete inhibition of LDH release.

DETAILED DESCRIPTION

The present invention provides binding molecules comprising a first binding domain capable of binding to a glycosylated staphylococcal surface protein, and a second binding domain capable of binding to a staphylococcal leukotoxin. Accordingly, the binding molecules are at least bi-specific in that they are capable of specifically binding to at least two different staphylococcus virulence factors. Preferably, the binding molecules are human binding molecules.

In one embodiment, the first binding domain and the second binding domain of the binding molecule comprise an antibody (or fragment thereof), an alternative scaffold, or a combination thereof (e.g., the binding molecule comprises an antibody (or fragment thereof) and an alternative scaffold, each of which binds to a different bacterial virulence factor). Methods of producing these binding molecules are also provided.

Preferably, these multi-specific binding molecules exhibit bactericidal activity against staphylococci and, thus, can be used in the prevention, treatment, detection, diagnosis and/or amelioration of a Staphylococcus infection, including MRSA and MSSA infections.

Prior to disclosing the invention in detail the following definitions are provided. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

The term “binding molecule” in the sense of the present disclosure indicates any molecule capable of specifically binding to, interacting with, or recognizing two or more different antigens or targets. In this sense, the binding molecule of the invention is at least “bispecific,” which term as used herein refers to a molecule that comprises at least a first binding domain that is capable of binding to one antigen or target, and a second binding domain that is capable of binding to another antigen or target. In particular, the binding molecule of the present invention is capable of binding to, interacting with and/or recognizing a staphylococcal leukotoxin and a glycosylated staphylococcal surface protein. The binding molecule of the invention also includes multispecific binding molecules that are capable of binding two or more antigens simultaneously, including, but not limited to, trispecific binding molecules having three binding domains, tetraspecific molecular having four binding domains, etc. Each binding domain of the multispecific binding molecules is capable of binding a different antigen or bacterial protein, e.g., different staphylococcus antigens or proteins.

According to the present invention, exemplary binding molecules are polypeptides. Such polypeptides may include proteinaceous parts and non-proteinaceous parts (e.g., chemical linkers or chemical cross-linking agents).

The term “binding domain” characterizes, in connection with the present disclosure, a domain of the binding molecule which is capable of specifically binding to/interacting with a given target epitope or a given target site on the target molecules, for example, a staphylococcal leukotoxin (e.g., LukAB, LukD and/or LukE) and/or a glycosylated staphylococcal surface protein (e.g., an SdgB-glycosylated staphylococcal SDR-containing protein).

Binding domains can be derived from a binding domain donor such as for example an antibody, protein A, ImmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain, an ankyrin consensus repeat domain or thioredoxin (Skerra, A., “Alternative Non-Antibody Scaffolds for Molecular Recognition,” Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., “A New Generation of Protein Display Scaffolds for Molecular Recognition,” Protein Sci. 15:14-27 (2006); Nicaise et al., “Affinity transfer by CDR grafting on a Nonimmunoglobulin Scaffold,” Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, “Scaffolds for Engineering Novel Binding Sites in Proteins,” Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated by reference in their entirety)). It is envisioned that a binding domain of the present invention comprises at least said part of any of the aforementioned binding domains that is required for binding to/interacting with a given target epitope or a given target site on the target molecules.

It is envisaged that the binding domain of the aforementioned binding domain donors is characterized by that part of these donors that is responsible for binding the respective target, i.e., when that part is removed from the binding domain donor, said donor loses its binding capability. “Loses” means a reduction of at least 50% of the binding capability when compared with the binding donor. Methods to map these binding sites are well known in the art—it is therefore within the standard knowledge of the skilled person to locate/map the binding site of a binding domain donor and, thereby, to “derive” said binding domain from the respective binding domain donors.

The first and/or second binding domains of the binding molecule may comprise an immunoglobulin, for example an antibody, fragment thereof, or derivative thereof. The first and/or second binding domains of the binding molecule may alternatively comprise an alternative scaffold. In one embodiment, the binding molecule may comprise one or more immunoglobulin binding domains and one or more alternative scaffold binding domains.

As used herein, the term “alternative scaffold” refers to a single chain polypeptidic framework typically of reduced size (e.g., less than about 200 amino acids) that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions. These scaffolds are based either on a conventional Ig backbone, or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted protein. For example, such a scaffold can be derived from Protein A, in particular, the Z-domain thereof (affibodies), ImmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain, an ankyrin consensus repeat domain, or thioredoxin (Skerra, A., “Alternative Non-Antibody Scaffolds for Molecular Recognition,” Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., “A New Generation of Protein Display Scaffolds for Molecular Recognition,” Protein Sci. 15:14-27 (2006); Nicaise et al., “Affinity Transfer by CDR Grafting on a Nonimmunoglobulin Scaffold,” Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, “Scaffolds for Engineering Novel Binding Sites in Proteins,” Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated by reference in their entirety). The structure of alternative scaffolds vary, but preferably are of human origin for those developed as therapeutics.

Non-Ig based scaffolds include, but are not limited to, lipocalins (used in “anticalins”), ankyrin repeat (AR) proteins (used in “designed AR proteins” or “DARPins”), fibronectin domain derivatives (used in “adnectin”), and avidity multimers (also known as “avimers”).

Anticalins, an engineered protein scaffold comprising a lipocalin backbone, are a suitable non-Ig based alternative scaffolds for use in the binding molecules of the present invention. Lipocalins, a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids, are the parental protein structure of anticalins. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel b-barrels. These proteins contain four exposed loops built on the rigid beta-barrel structure. Exemplary lipocalins have a domain size of about 160 amino acids to about 180 amino acids and have been developed to contain randomization of 16 accessible amino acids within the four exposed loops.

Proteins comprising ankyrin repeat (AR) proteins are another suitable non-Ig based alternative scaffold for use in the binding molecules of the present invention. AR proteins comprise a 33 amino acid protein motif consisting of two alpha helices separated by loops, which repeats mediate protein-protein interactions. Designed Ankyrin Repeat Proteins (DARPins) comprise an engineered protein scaffold resulting from rational design strategies (e.g., multiple sequence alignments and statistical analysis) based on human AR proteins. DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions. DARPin libraries are preferentially screened using ribosome display, and library members typically are well produced in E. coli, do not aggregate, and display high thermodynamic stability. Preferably, DARPins contain two to four of these motifs flanked by N- and C-terminal capping motifs to shield hydrophobic regions and allow increased solubility.

Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable non-Ig based alternative scaffold. For example, the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a beta-sandwich with seven beta-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges. In one aspect, the connecting loops of FN10, each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties. Adnectin (Adnexus, now Bristol-Myers-Squibb) is an exemplary scaffold generated using 10^(th) FN3 domains randomized and displayed in this way. An alternative exemplary scaffold comprising FN3 domains is a Centyrin™. Centryrins™ contain the consensus sequence of FN3 domains of human Tenascin C (TNC). Centyrin™ scaffolds have loops (i.e., DE, BC and FG) that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues. These molecules have excellent biophysical properties (e.g., greater than 100 mg/mL expression, greater than 170 mg/mL solubility, greater than 82° C. melting temperature, low predicted immunogenicity, and stable in serum for more than one month), and can be engineered for improved stability. Therefore, Centyrins™ are a suitable alternative scaffold derived from FN domains for use in the present binding molecules.

In one embodiment, the binding molecule as described herein comprises a Centyrin™ or FN3 domain that comprises an amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666. Exemplary FN3 domain sequences of the binding molecule described herein include Luk17 and Luk12 (SEQ ID NO:14 and SEQ ID NO:22, respectively).

The avimer structure can also be used as a protein backbone to generate a suitable non-Ig based alternative scaffold. The avimer scaffold is based on oligomerization of A-domains from low-density lipoprotein (LRL) cell surface receptors. Preferably, the avimer scaffold comprises universally conserved residues of the A-domain, which is about 35 amino acids in length and comprises four loops with three disulfide bridges.

Additionally, the present invention also contemplates binding molecules comprising an Ig-like scaffold selected from, e.g., a nanobody, a domain antibody (dAb), a bispecific T cell engager (BiTE), a dual affinity retargeting protein (DART), and a Tetravalent tANDem AntiBodies (TandAb) (see, e.g., Wurch et al., “Novel Protein Scaffolds as Emerging Therapeutic Proteins: From Discovery to Clinical Proof-of-Concept,” Trends in Biotechnology 30(11):575-582 (2012), which is hereby incorporated by reference in its entirety).

The binding molecule can also be a fusion protein comprising an antibody, fragment thereof, or derivative linked to an alternative scaffold. In one embodiment, binding molecules comprise an antibody, a fibronectin domain or an antibody-fibronectin domain fusion protein. For example, in one aspect, the binding molecule of the present invention comprises a full-length antibody or antibody fragment that is capable of binding to a glycosylated staphylococcal surface protein, such as a SdgB-glycosylated SDR-containing protein, and further comprises an alternative scaffold that is capable of binding to a staphylococcal leukotoxin, such as LukAB, LukD, or LukE. It is understood that clinical isolates of Staph may have varying sequences among different strains. Therefore, in one aspect, the alternative scaffold comprises a FN3 domain that is capable of binding to LukA having an amino acid sequence with 80%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:10, LukB having an amino acid sequence with 80%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:11, LukD having an amino acid sequence with 80%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:12, or LukE having an amino acid sequence with 80%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:13. In one aspect, the alternative scaffold of the binding molecule comprises a FN3 domain that is capable of binding to LukA having the amino acid sequence of SEQ ID NO:10, LukB having the amino acid sequence of SEQ ID NO:11, LukD having the amino acid sequence of SEQ ID NO:12, or LukE having the amino acid sequence of SEQ ID NO:13. In another aspect, the alternative scaffold of the binding molecule comprises a FN3 domain that is capable of binding a LukAB complex comprising LukA having the amino acid sequence of SEQ ID NO:10 and LukB having the amino acid sequence of SEQ ID NO:11. In another aspect, the alternative scaffold of the binding molecule comprises a FN3 domain that is capable of binding a LukED complex comprising LukD having the amino acid sequence of SEQ ID NO:12, and LukE having the amino acid sequence of SEQ ID NO:13.

It is envisaged that the binding molecule is produced by (or obtainable by) phage-display or library screening methods. Alternatively, the binding molecule is produced by grafting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold, for example, a scaffold as disclosed herein.

The terms “capable of binding to”, “binding to”, “specifically recognizing”, “directed to” and “reacting with” mean, in accordance with this invention, that a binding domain is capable of specifically interacting with one or more amino acids of an epitope.

The term “is not capable of specific binding” means that a binding domain of the present invention does not bind another protein or antigen, e.g., a non-target protein, under the conditions tested. For example, in the present disclosure, Protein A binding was measured using plate-based direct ELISA assays under the conditions specified in the Examples, which detected no appreciable binding to Protein A for binding molecules containing the CH3 mutations H435R/Y436F (e.g., constructs 3-7). However, if the same experiments were performed with a 100-1000-fold excess of protein, some Protein A binding (non-specific) may be detected. This general principle also applies to human FcγRI binding (or lack thereof) and Sbi binding (or lack thereof) under the conditions provided herein.

“Proteins” (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term “polypeptide” as used herein is a chain of more than 30 amino acids coupled together. Polypeptides may form multimers such as dimers, trimers and higher oligomers, i.e., consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains.

The terms “polypeptide” and “protein” also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g., by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. A “polypeptide” when referred to herein may also be chemically modified such as pegylated. Such modifications are well known in the art.

The term “amino acid” or “amino acid residue” typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

The term “virulence factor” as used herein refers to a molecule expressed by bacteria that enables the bacteria to achieve colonization of a niche in the host (including adhesion to cells), immunoevasion (i.e., evasion of the host's immune response), immunosuppression (i e, inhibition of the host's immune response), entry into and exit out of cells (if the pathogen is an intracellular one), and/or obtain nutrition from the host. The virulence factors may be encoded on mobile genetic elements, such as bacteriophages, and can easily be spread through horizontal gene transfer. Non-limiting examples of Staphylococcus aureus virulence factors include hyaluronidase, protease, coagulase, lipases, deoxyribonucleases, enterotoxins and other toxins, e.g., toxins that act on cell membranes such as LukAB, LukD or LukE. For the purposes of this invention, staphylococcal surface proteins, such as SDR-containing proteins, e.g., ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG and SdrH, are also considered to be a virulence factors.

The definition of the term “antibody” includes embodiments such as monoclonal, chimeric, single chain, humanized and human antibodies. In addition to full-length antibodies, the definition also includes antibody derivatives and antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab′)₂, Fv, scFv fragments or single domain antibodies such as domain antibodies or nanobodies, single variable domain antibodies or immunoglobulin single variable domains comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains; see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring harbor Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Kontermann and Dübel, ANTIBODY ENGINEERING, Springer, 2nd ed. 2010; and Little, RECOMBINANT ANTIBODIES FOR IMMUNOTHERAPY, Cambridge University Press 2009, each of which is hereby incorporated by reference in its entirety. Said term also includes diabodies or Dual-Affinity Re-Targeting (DART) antibodies. Further envisaged are (bispecific) single chain diabody, tandem diabody (Tandab), “minibodies” exemplified by a structure which is as follows: (VH-VL-CH3)₂, (scFv-CH3)₂ or (scFv-CH3-scFv)₂, “Fc DART” and “IgG DART”, multibodies such as triabodies.

Immunoglobulin single variable domains encompass not only an isolated antibody single variable domain polypeptide, but also larger polypeptides that comprise one or more monomers of an antibody single variable domain polypeptide sequence.

Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778 to Ladner et al., Kontermann and Dübel, ANTIBODY ENGINEERING, Springer, 2^(nd) ed. 2010, and Little, RECOMBINANT ANTIBODIES FOR IMMUNOTHERAPY, Cambridge University Press, 2009, each of which is hereby incorporated by reference in its entirety) can be adapted to produce single chain antibodies specific for elected polypeptide(s). Also, transgenic animals may be used to express humanized antibodies specific for polypeptides and fusion proteins of this invention. For the preparation of monoclonal antibodies, any technique, providing antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Köhler and Milstein, “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256: 495-497 (1975), which is hereby incorporated by reference in its entirety), the trioma technique, the human B cell hybridoma technique (Kozbor et al., “The Production of Monoclonal Antibodies From Human Lymphocytes,” Immunology Today 4:72-79 (1983), which is hereby incorporated by reference in its entirety) and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96 (1985), which is hereby incorporated by reference in its entirety). Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of a target polypeptide, such as CD3 epsilon (Schier et al., “Efficient In Vitro Affinity Maturation of Phage Antibodies Using BIAcore Guided Selections,” Human Antibodies Hybridomas 7:97-105 (1996); Malmborg et al., “BIAcore as a Tool in Antibody Engineering,” J. Immunol. Methods 183:7-13 (1995), each of which is hereby incorporated by reference in its entirety). It is also envisaged in the context of this invention that the term “antibody” comprises antibody constructs, which may be expressed in a host as described herein below, e.g., antibody constructs which may be transfected and/or transduced via, inter alia, viruses or plasmid vectors.

Furthermore, the term “antibody” as employed herein also relates to derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of “antibody variants” include humanized variants of non-human antibodies, “affinity matured” antibodies (see e.g., Hawkins et al., “Selection of Phage Antibodies by Binding Affinity. Mimicking Affinity Maturation,” J. Mol. Biol. 254:889-896 (1992) and Lowman et al., “Selecting High-Affinity Binding Proteins by Monovalent Phage Display,” Biochemistry 30:10832-10837 (1991), each of which is hereby incorporated by reference in its entirety) and antibody mutants with altered effector function(s) (see, e.g., U.S. Pat. No. 5,648,260 to Winter et al., Kontermann and Dübel, ANTIBODY ENGINEERING, Springer, 2^(nd) ed. 2010, and Little, RECOMBINANT ANTIBODIES FOR IMMUNOTHERAPY, Cambridge University Press, 2009, each of which is hereby incorporated by reference in its entirety).

One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in U.S. Pat. No. 5,223,409 to Ladner et al.; Smith, “Filamentous Fusion Phage: Novel Expression Vectors That Display Cloned Antigens on the Virion Surface,” Science 228:1315-1317 (1985); Clackson et al., “Making Antibody Fragments Using Phage Display Libraries,” Nature 352:624-628 (1991), each of which is hereby incorporated by reference in its entirety.

In addition to the use of display libraries, the specified antigen can be used to immunize a non-human animal, e.g., a rodent, a mouse, hamster, or rat. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al., “Antigen-Specific Human Monoclonal Antibodies From Mice Engineered With Human Ig Heavy and Light Chain YACs,” Nature Genetics 7:13-21 (2004), US 2003-0070185 to Jakobovits et al., WO96/34096 to Kucherlapati et al., and WO96/33735 to Kucherlapati et al., each of which is hereby incorporated by reference in its entirety.

An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or “deimmunization” by the methods disclosed in WO98/52976 to Carr et al. and WO00/34317 to Can et al., which are hereby incorporated by reference in their entirety. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined in WO98/52976 to Can et al. and WO00/34317 to Can et al., both of which are hereby incorporated by reference in their entirety). For detection of potential T cell epitopes, a computer modeling approach termed “peptide threading” can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO98/52976 to Can et al. and WO00/34317 to Can et al., both of which are hereby incorporated by reference in their entirety. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. Human germline sequences, e.g., are disclosed in Tomlinson et al., “The Repertoire of Human Germline VH Sequences Reveals About Fifty Groups of VH Segments With Different Hypervariable Loops,” J. Mol. Biol. 227:776-798 (1992); Cook et al., “The Human Immunoglobulin VH Repertoire,” Immunol. Today 16 (5):237-242 (1995); and Tomlinson et al., “The Structural Repertoire of the Human V Kappa Domain,” EMBO J. 14:14:4628-4638 (1995), each of which is hereby incorporated by reference in its entirety. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, L A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Pat. No. 6,300,064 to Knappik et al., which is hereby incorporated by reference in its entirety. The pairing of a VH and VL together forms a single antigen-binding site. The constant heavy (CH) domain most proximal to VH is designated as CH1. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for specific interactions of the antibody with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are referred to as H1, H2, and H3, while CDR constituents on the light chain are referred to as L1, L2, and L3.

The term “variable” refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the “variable domain(s)” or “V-region”). Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions.

These sub-domains are called “hypervariable” regions or “complementarity determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the “framework” regions (FRM). The variable domains of naturally occurring heavy and light chains each comprise four FRM regions, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site (see Wu and Kabat, “An Analysis of the Sequences of the Variable Regions of Bence Jones Proteins and Myeloma Light Chains and their Implications for Antibody Complementarity,” J. Exp. Med. 132:211-250 (1970), which is hereby incorporated by reference in its entirety). The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as, for example, antibody-dependent, cellular cytotoxicity and complement activation.

The term “hypervariable region” (also known as “complementarity determining regions” or CDRs) when used herein refers to the amino acid residues of an antibody which are (usually three or four short regions of extreme sequence variability) within the V-region domain of an immunoglobulin which form the antigen-binding site and are the main determinants of antigen specificity. There are at least two methods for identifying the CDR residues: (1) An approach based on cross-species sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991), which is hereby incorporated by reference in its entirety); and (2) An approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901-917 (1987), which is hereby incorporated by reference in its entirety). However, to the extent that two residue identification techniques define regions of overlapping, but not identical regions, they can be combined to define a hybrid CDR. However, in general, the CDR residues are preferably identified in accordance with the Kabat (numbering) system.

The terms “antigen-binding domain”, “antigen-binding fragment” and “antibody binding region” when used herein refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the “epitope” as described herein above. As mentioned above, an antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1) a Fab fragment, a monovalent fragment having the VL, VH, constant light (CL) and CH1 domains; (2) a F(ab′)₂ fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having the two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which is hereby incorporated by reference) which has a VH domain; (6) an isolated complementarity determining region (CDR), and (7) a single chain Fv (scFv), for example, derived from a scFV-library. Although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (see e.g., Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), which is hereby incorporated by reference in its entirety). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.), each of which is hereby incorporated by reference in its entirety.

The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., “Making Antibody Fragments Using Phage Display Libraries,” Nature 352:624-628 (1991) and Marks et al., “By-Passing Immunization. Human Antibodies From V-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597 (1991), for example, each of which is hereby incorporated by reference in its entirety.

The monoclonal antibodies of the present invention specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., “Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains With Human Constant Region Domains,” Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984), both of which are hereby incorporated by reference in their entirety). Chimeric antibodies of interest herein include “primitized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

A monoclonal antibody can be obtained from a non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., “Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains With Human Constant Region Domains,” Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1985); Takeda et al., “Construction of Chimeric Processed Immunoglobulin Genes Containing Mouse Variable and Human Constant Region Sequences,” Nature 314:452 (1985); U.S. Pat. No. 4,816,567 to Cabilly et al.; U.S. Pat. No. 4,816,397 to Boss et al.; EP 0171496 to Tanaguchi et al.; EP 0173494 to Morrison et al., GB 2177096 to Neuberger et al., each of which are hereby incorporated by reference in their entirety.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, “humanized antibodies” as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:522-525 (1986) and Reichmann et al., “Reshaping Human Antibodies for Therapy,” Nature 332:323-329 (1988) each of which is hereby incorporated by reference in its entirety.

Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539 to Winter et al., which is hereby incorporated by reference in its entirety). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

Humanized antibodies or fragments thereof can, for example, be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison, S L., “Transfectomas Provide Novel Chimeric Antibodies,” Science 229:1202-1207 (1985); by U.S. Pat. No. 5,585,089 to Queen et al.; U.S. Pat. No. 5,693,761 to Queen et al.; U.S. Pat. No. 5,693,762 to Queen et al.; U.S. Pat. No. 5,859,205 to Adair et al.; and U.S. Pat. No. 6,407,213 to Carter et al., each of which is hereby incorporated by reference in its entirety. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

A humanized antibody can be optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or back mutations. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., “Construction and Testing of Mouse—Human Heteromyelomas for Human Monoclonal Antibody Production,” Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312 (1983); Kozbor et al., “The Production of Monoclonal Antibodies from Human Lymphocytes,” Immunology Today 4:72-79 (1983); Olsson et al., “Human—Human Monoclonal Antibody-Producing Hybridomas: Technical Aspects,” Meth. Enzymol. 92:3-16 (1982), each of which is hereby incorporated by reference in its entirety), and may be made according to the teachings of EP239400 to Winter, which is hereby incorporated by reference in its entirety.

The term “human antibody” includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991), which is hereby incorporated by reference in its entirety. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3. The human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.

A “bispecific” or “bifunctional” antibody or immunoglobulin is an artificial hybrid antibody or immunoglobulin having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments (see e.g., Songsivilai & Lachmann, “Bispecific Antibody: A Tool for Diagnosis and Treatment of Disease,” Clin. Exp. Immunol. 79:315-321 (1990), which is hereby incorporated by reference in its entirety). Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Pat. No. 4,816,567 to Cabilly, which is hereby incorporated by reference in its entirety). Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler et al., “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256:495 (1975), which is hereby incorporated by reference in its entirety) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.

The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called “hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996)), each of which is hereby incorporated by reference in its entirety.

Typically, CDRs form a loop structure that can be classified as a canonical structure. The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” Nature 342:877 (1989); Martin and Thornton, “Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies,” J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues.

The term “canonical structure” may also include considerations as to the linear sequence of the antibody, for example, as catalogued by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991), which is hereby incorporated by reference in its entirety. The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner and is the preferred scheme applied in the present invention as also mentioned elsewhere herein.

Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al and/or revealed by other techniques, for example, crystallography and two or three-dimensional computational modeling.

Accordingly, a given antibody sequence may be placed into a canonical class which allows for, among other things, identifying appropriate chassis sequences (e.g., based on a desire to include a variety of canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987), which is hereby incorporated by reference in its entirety, and their implications for construing canonical aspects of antibody structure, are described in the literature.

CDR3 is typically the greatest source of molecular diversity within the antibody-binding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988, which is hereby incorporated by reference in its entirety. One of skill in the art will recognize that each subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the antigen, i.e., the antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to the Kabat CDRs, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al., which is hereby incorporated by reference in its entirety.

Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia (see e.g., Chothia et al., “Structural Repertoire of the Human VH Segments,” J. Mol. Biol. 227:799-817 (1987); and Tomlinson et al., “The Structural Repertoire of the Human V Kappa Domain,” EMBO J. 14: 4628-4638 (1995), each of which is hereby incorporated by reference in its entirety). Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual, Duebel and Kontermann (eds.), Springer-Verlag, which is hereby incorporated by reference in its entirety). Embodiments described with respect to Kabat CDRs can alternatively be implemented using similar described relationships with respect to Chothia hypervariable loops or to the AbM-defined loops. The sequence of antibody genes after assembly and somatic mutation is highly varied, and these varied genes are estimated to encode 10¹⁰ different antibody molecules (Immunoglobulin Genes, 2^(nd) ed., eds. Jonio et al., Academic Press, 1995, which is hereby incorporated by reference in its entirety). Accordingly, the immune system provides a repertoire of immunoglobulins.

The term “repertoire” refers to at least one nucleotide sequence derived wholly or partially from at least one sequence encoding at least one immunoglobulin. The sequence(s) may be generated by rearrangement in vivo of the V, D, and J segments of heavy chains, and the V and J segments of light chains. Alternatively, the sequence(s) can be generated from a cell in response to which rearrangement occurs, e.g., in vitro stimulation. Alternatively, part or all of the sequence(s) may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e.g., U.S. Pat. No. 5,565,332 to Hoogenboom et al., which is hereby incorporated by reference in its entirety. A repertoire may include only one sequence or may include a plurality of sequences, including ones in a genetically diverse collection.

The term “framework region” refers to the art-recognized portions of an antibody variable region that exist between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for the presentation of the six CDRs (three from the heavy chain and three from the light chain) in three dimensional space, to form an antigen-binding surface.

The term “fibronectin type III (FN3) domain” (FN3 domain) as used herein refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, “Proposed Acquisition of an Animal Protein Domain by Bacteria,” Proc. Nat. Acad. Sci. USA 89:8990-8994 (1992); Meinke et al., “Cellulose-Binding Polypeptides From Cellulomonas fimi: Endoglucanase D (CenD), a Family A beta-1,4-Glucanase,” J. Bacteriol. 175:1910-1918 (1993); Watanabe et al., “Gene Cloning of Chitinase A1 From Bacillus circulans WL-12 Revealed its Evolutionary Relationship to Serratia chitinase and to the Type III Homology Units of Fibronectin,” J. Biol. Chem. 265:15659-15665 (1990), each of which is hereby incorporated by reference in its entirety). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. Publ. No. 2010/0216708 to Jacobs et al., which is hereby incorporated by reference in its entirety. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3^(rd) FN3 domain of tenascin (TN3), or the 10^(th) FN3 domain of fibronectin (FN10).

The term “substituting” or “substituted” or “mutating” or “mutated” as used herein refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

The term “randomizing” or “randomized” or “diversified” or “diversifying” as used herein refers to making at least one substitution, insertion or deletion in a polynucleotide or polypeptide sequence.

“Tencon” as used herein refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO:1 and described in U.S. Pat. Publ. No. US2010/0216708 to Jacobs et al., which is hereby incorporated by reference in its entirety.

The term “library” refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.

The term “vector” means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

A vector can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the vector. Thus, a “vector,” as used herein, refers broadly to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.

The present application generally relates to multi-specific binding molecules that bind to at least two different bacterial virulence factors, methods for producing these binding molecules, and the use of these binding molecules to detect or diagnose, prevent and/or treat bacterial infections. In particular, the binding molecules comprise at least two binding domains, for example, an antibody or antibody fragment and an alternative scaffold. The first binding domain is capable of binding to a glycosylated staphylococcal surface protein, preferably an SdgB glycosylated SDR-containing protein. The second binding domain is capable of binding to a staphylococcal leukotoxin, preferably LukAB, LukD or LukE. These multi-specific binding molecules have neutralizing activity against the protein antigen they bind, i.e., once the binding molecule binds to its target staphylococcal proteins, the activity of those proteins is substantially, or completely eliminated. Accordingly, the multi-specific binding molecules also have neutralizing or bactericidal activity against staphylococcus itself, and, thus, can be used in the treatment and/or amelioration of a Staphylococcus infection, including methicillin-resistant and methicillin-sensitive Staphylococcus aureus.

The binding molecules hereof may also be capable of specifically binding to one or more fragments of staphylococci (and other gram-positive and/or gram-negative bacteria) such as, inter alia, a preparation of one or more proteins and/or (poly)peptides derived from staphylococci or one or more recombinantly produced staphylococci proteins and/or polypeptides. For methods of treatment and/or prevention of staphylococcal infections the binding molecules are preferably capable of specifically binding to surface accessible proteins of staphylococci. Surface proteins of staphylococci include, but are not limited to, clumping factor (Clf)A and ClfB, SDR-proteins (e.g., SdrC, SdrD, SdrE, SrdF, SdrG and SdrH), Protein A, Sbi, fibronectin-binding protein (FnbA) and serine-rich adhesin for platelets (SraP). In S. aureus and S. epidermidis, SDR domains of all SDR-containing proteins are heavily glycosylated by at least two glycosyltransferases, SdgA and SdgB, which prevents degradation of these proteins by host proteases, thereby preserving bacterial host tissue interactions. These sugar modifications also represent a dominant antibody epitope. Preferably, the binding molecules bind to glycosylated surface staphylococcal proteins, in particular SdgB glycosylated SDR proteins (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog. 9(10):e1003653 (2013), which is hereby incorporated by reference in its entirety).

As described herein, the binding molecules hereof can comprise intact immunoglobulin molecules such as polyclonal or monoclonal antibodies or antigen-binding fragments including, but not limited to, Fab, F(ab′), F(ab′)₂, Fv, dAb, Fd, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, and (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to staphylococci or a fragment thereof. In certain embodiments the binding molecules hereof are human monoclonal antibodies.

In one aspect, the first binding domain is a full-length antibody or antibody fragment. Preferably, the full-length antibody or antibody fragment is resistant to proteolytic degradation by a staphylococcal protease that cleaves wild-type IgG1 (such as the staphylococcal protease, Staphylococcus aureus V8 protease, that cleaves wild-type IgG1 between or at residues 222-237 (EU numbering) within SEQ ID NO:60). In another aspect, the full-length antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

In one aspect, the first binding domain comprises an immunoglobulin heavy chain variable (VH) region having the amino acid sequence selected from the group of VH region amino acid sequences of SEQ ID NOs:60, 62, 64 or 66. In another aspect, the first binding domain comprises an immunoglobulin light chain variable (VL) region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. Alternatively, the first binding domain comprises (a) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (b) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. In on embodiment, the first binding domain comprises (1) a VH region having the amino acid sequence of SEQ ID NO:60, and a VL region having the amino acid sequence of SEQ ID NO:61; (2) a VH region having the amino acid sequence of SEQ ID NO:62, and a VL region having the amino acid sequence of SEQ ID NO:63; (3) a VH region having the amino acid sequence of SEQ ID NO:64, and a VL region having the amino acid sequence of SEQ ID NO:65; (4) a VH region having the amino acid sequence of SEQ ID NO:66, and a VL region having the amino acid sequence of SEQ ID NO:67; (5) a VH region having the amino acid sequence of SEQ ID NO:68, and a VL region having the amino acid sequence of SEQ ID NO:69; (6) a VH region having the amino acid sequence of SEQ ID NO:70, and a VL region having the amino acid sequence of SEQ ID NO:71; (7) a VH region having the amino acid sequence of SEQ ID NO:72, and a VL region having the amino acid sequence of SEQ ID NO:73; (8) a VH region having the amino acid sequence of SEQ ID NO:74, and a VL region having the amino acid sequence of SEQ ID NO:75; (9) a VH region having the amino acid sequence of SEQ ID NO:76, and a VL region having the amino acid sequence of SEQ ID NO:77; or (10) a VH region having the amino acid sequence of SEQ ID NO:78, and a VL region having the amino acid sequence of SEQ ID NO:79.

The binding molecules described herein comprise a second binding domain that is capable of specifically binding to other staphylococcal molecules including, for example, bicomponent toxins. In addition to PVL (also known as leukocidin S/F-PV or LukSF-PV) and gamma-hemolysin (HlgAB and HlgCB), the repertoire of bi-component leukotoxins produced by S. aureus is known to include leukocidin E/D (“LukED”), leukocidin A/B (“LukAB”) and leukocidin M/F (“LukMF”). Thus, the S-class subunits of these bi-component leukocidins include HlgA, HlgC, LukE, LukS-PV, LukA, and LukM, and the F-class subunits include HlgB, LukD, LukF-PV, LukB, and LukF′-PV. The S. aureus S- and F-subunits are not leukocidin-specific. That is, they are interchangeable such that other bi-component combinations could make a functional pore in a white blood cell, greatly increasing the repertoire of leukotoxins (Meyer et al., “Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding,” Infect. Immun. 77(1):266-273 (2009), the disclosure of which is hereby incorporated by reference in its entirety).

In one embodiment, the binding molecules of the present invention bind to one or more staphylococcal leukotoxins selected from leukotoxin A (LukA), leukotoxin B (LukB), leukotoxin AB (LukAB), leukotoxin D (LukD), leukotoxin E (LukE), leukotoxin ED (LukED), Panton-Valentine leukocidin S (LukS-PV), Panton-Valentine leukocidin F (LukF-PV), Panton-Valentine leukocidin (LukSF/PVL), gamma hemolysin A (HlgA), gamma hemolysin C (HlgC), gamma hemolysin B (HlgB), gamma hemolysin AB (HlgAB), and gamma-hemolysin BC (HlgBC). In one embodiment, the binding molecule binds to one or more of the staphylococcal leukotoxins selected from LukAB, LukD or LukE In another embodiment, the binding molecules hereof are capable of specifically binding to a fragment of the above-mentioned proteins, wherein the fragment at least comprises an antigenic determinant recognized by the binding molecules hereof. An “antigenic determinant” as used herein, is a moiety that is capable of binding to a binding molecule hereof with sufficiently high affinity to form a detectable antigen-binding molecule complex. In another embodiment, the binding molecules hereof are capable of specifically binding to a fragment of the above-mentioned staphylococcal leukotoxin proteins containing a neutralizing epitope. Binding to a neutralizing epitope of the leukotoxin protein eliminates or substantially eliminates the activity of the protein.

In one aspect, the second binding domain that is capable of binding a staphylococcal leukotoxin is an alternative scaffold. Preferably, the second binding domain comprises a fibronectin type III (FN3) domain, e.g., an isolated, recombinant and/or synthetic protein scaffold based on a consensus sequence of fibronectin type III (FN3) repeat protein. The FN3 scaffolds of the present invention offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E. coli allowing for increased expression of protein as a function of resources versus mammalian cell expression, ability to be engineered into bispecific molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues.

Moreover, the FN3 scaffolds possess many of the properties of antibodies in relation to their fold that mimics the variable region of an antibody. For example, the orientation of FN3 domains enables the FN3 loops to be exposed similar to antibody complementarity determining regions (CDRs). The loop regions are able to bind to cellular targets and can be altered, e.g., affinity matured, to improve certain binding or related properties.

In one embodiment, the binding molecule described herein comprises one or more FN3 domains. In one aspect, the FN3 domain of the binding molecule is derived from the non-naturally occurring FN domain of Tencon (SEQ ID NO:1). Tencon, like other FN3 domains, has a beta-sandwich structure with seven beta-strands, referred to as A, B, C, D, E, F, and G, linked by six loops, referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc. Natl. Acad. Sci. USA 89:8990-8992 (1992) and U.S. Pat. No. 6,673,901 to Koide et al., which are hereby incorporated by reference in their entirety). Three of the six loops, i.e., BC, DE, and FG loops, are at the top of the FN3 domain, and three of the loops, i.e., AB, CD, and EF loops, are at the bottom of the FN3 domain. These loops span at or about amino acid residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO:1. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 of SEQ ID NO:1 are altered for binding specificity and affinity in the binding molecule described herein. In one aspect, the binding molecules described herein include one or more binding domains that comprise an alternative scaffold comprising at least one, at least two, or all three loops at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and/or 75-81 of SEQ ID NO:1. In one aspect, the binding molecules include one or more binding domains that comprise an alternative scaffold comprising at least one, at least two, or all three loop regions at or about residues 22-28, 51-54, and 75-81 of SEQ ID NO:1. One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library, and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.

As discussed above, FN3 domains contain two sets of CDR-like loops (i.e., BC, DE, and FG loops and AB, CD, and EF loops) on the opposite faces of the molecule. The two sets of loops are separated by beta-strands that form the center of the FN3 structure. Like the loops, these beta-strands can be altered to enhance binding specificity and affinity. Preferably, some or all of the surface exposed residues in the beta strands are randomized without affecting (or minimally affecting) the inherent stability of the scaffold. One or more of the beta-strands can interact with a target protein. The beta-strands in a scaffold provide a flat binding surface (compared to a curved binding surface found in protein scaffolds containing adjacent loops) that effects the target proteins, or specific epitopes on those target proteins, that can be bound effectively by the scaffold. In one aspect, the binding molecules of the present invention includes a binding domain that comprises an alternative scaffold comprising one or more beta-strands that bind to a bacterial virulence factor, e.g., a Staphylococcal leukotoxin.

In one aspect, the second binding domain of the binding molecule comprises a FN3 domain that binds to LukA having the amino acid sequence of SEQ ID NO:10, LukB having the amino acid sequence of SEQ ID NO:11, LukD having the amino acid sequence of SEQ ID NO:12, or LukE having the amino acid sequence of SEQ ID NO:13. Alternatively, the second binding domain of the binding molecule comprises a FN3 domain that binds to a LukAB complex comprising LukA having the amino acid sequence of SEQ ID NO:10 and LukB having the amino acid sequence of SEQ ID NO:11 and/or a LukED complex comprising LukE having the amino acid sequence of SEQ ID NO:13 and LukD having the amino acid sequence of SEQ ID NO:12.

In one aspect, the second binding domain of the binding molecule competes with an FN3 domain having an amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666 for binding to (i) LukA having the amino acid sequence of SEQ ID NO:10; (ii) LukB having the amino acid sequence of SEQ ID NO:11; (iii) LukD having the amino acid sequence of SEQ ID NO:12; and/or (iv) LukE having the amino acid sequence of SEQ ID NO:13.

In yet another aspect, the binding molecule comprises (a) a first binding domain that comprises (i) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (ii) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67; and (b) a second binding domain that comprises the amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666. In one embodiment, the second binding domain comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:22.

In another aspect, the binding molecule comprises one or more additional binding domains, e.g., third, fourth, fifth, etc. binding domains. The additional binding domain(s) are capable of binding to a different glycosylated staphylococcal surface protein than is bound by the first binding domain of the binding molecule, and are capable of binding to a different staphylococcal leukotoxin that is bound by the second binding domain of the binding molecule. In other words, each binding domain of the binding molecule binds to a unique antigen. The additional binding domains of the binding molecule may be immunoglobulin domains, alternative scaffold domains, or a combination thereof.

In one aspect, the binding molecule does not exhibit non-specific binding to other staphylococcal or host (e.g., human) protein antigens. For example, in one aspect, the binding molecule is not capable of specific binding to staphylococcal Protein A or second binding protein for immunoglobulins (Sbi). In another aspect, the binding molecule is not capable of specific binding to FcγRI, in particular human FcγRI.

In another aspect, the binding molecule does retain specific binding capacity to FcRn.

The binding molecules of the present disclosure may incorporate other subunits, e.g., via covalent interaction. All or a portion of an antibody constant region may be attached to the binding molecule to impart antibody-like properties, e.g., complement activity (CDC), half-life, etc. For example, effector function can be provided and/or controlled, e.g., by modifying Clq binding and/or FcγR binding and thereby changing complement dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC) activity.

“Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., protein scaffold loops) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.). Additionally, a toxin conjugate, albumin or albumin binders, polyethylene glycol (PEG) molecules may be attached to the binding molecule for desired properties. Any of these fusions may be generated by standard techniques, for example, by expression of the binding molecule from a recombinant fusion gene constructed using publically available gene sequences.

The binding molecules of the present disclosure can be used as monospecific in monomeric form or as bi- or multi-specific (for different protein targets or epitopes on the same protein target) in multimer form. The attachments may be covalent or non-covalent. For example, a dimeric bispecific binding molecule has one subunit with specificity for a first target protein or epitope and a second subunit with specificity for a second target protein or epitope. Binding molecule subunits can be joined in a variety of conformations that can increase the valency and thus the avidity of antigen binding.

The binding molecules of the present disclosure may also comprise linker peptides, which are known in the art to connect a protein or polypeptide domain with a biologically active peptide, producing a fusion protein. The linker peptides can be used to make a functional fusion protein from two unrelated proteins that retain the activities of both proteins. For example, an FN3 domain which specifically binds to a leukotoxin can be fused by means of a linker to an antibody, antibody fragment, scFv, or peptide ligand capable of binding to a staphylococcal surface protein to form a fusion protein which binds to the staphylococcal surface protein and neutralizes the harmful leukotoxin effects. Fusion proteins can be produced by art-recognized cloning techniques. For example, an oligonucleotide encoding the linker peptide is ligated between the genes encoding the domains of interest to form one fused gene encoding the entire single-chain protein. The 5′ end of the linker oligonucleotide is fused to the 3′ end of the first gene, and the 3′ end of the linker is fused to the 5′ end of the second gene. The entire fused gene can be transfected into a host cell by means of an appropriate expression vector.

Linker peptides of the present invention can vary in length and amino acid composition. For example, a linker peptide of the invention may be from about 2 to about 50 amino acids in length, about 20 to about 45 amino acids in length, about 25 to about 40 amino acids in length, about 30 to about 35 amino acids in length, about 24 to about 27 amino acids in length, or about 20 amino acids in length.

Most linker peptides are composed of repetitive modules of one or more of the amino acids glycine and serine. Exemplary linker peptides include, e.g., (Gly-Gly)_(n), (Gly-Ser)_(n) and (Gly₃-Ser)_(n), wherein _(n) is an integer from 1-25. The length of the linker may be appropriately adjusted as long as it does not affect the function of the polypeptides. The standard 15 amino acid (Gly₄-Ser)₃ linker peptide has been well-characterized (e.g., within the context of an antibody single-chain Fv (scFv) domain) and has been shown to adopt an unstructured, flexible conformation. In addition, this linker peptide does not interfere with assembly and binding activity of the domains it connects (Freund et al., “Characterization of the Linker Peptide of the Single-Chain Fv Fragment of an Antibody by NMR Spectroscopy,” FEBS 320:97 (1993), the disclosure of which is hereby incorporated by reference in its entirety). Accordingly, a particularly effective linker for forming bifunctional fusion proteins comprises a Gly-Ser linker. In one aspect, the linker peptide contains the following amino acid sequence (SEQ ID NO:2):-G-G-G-G-S-G-G-G-G-S-G-G-G-G-S-G-G-G-G-S-.

However, the linker peptides of the invention also contemplate variant forms of this linker peptide, which can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the linker peptide such that one or more amino acid substitutions, additions or deletions are introduced into the linker peptide. For example, mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues such that the ability of the linker peptide is not altered. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a conservative amino acid substitution within the context of a peptide linker comprising a gly-ser motif (e.g., (Gly-Gly)_(n), (Gly-Ser)_(n) and (Gly₃-Ser)_(n)) would include substitution of the Gly and/or Ser residue with another uncharged polar side chains (i.e., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine).

The binding molecule of the present invention is preferably an “isolated” binding molecule. “Isolated” when used to describe the binding molecule disclosed herein, means a binding molecule that has been identified, separated and/or recovered from a component of its production environment. Preferably, the isolated binding molecule is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the binding molecule will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated binding molecule will be prepared by at least one purification step.

Amino acid sequence modifications of the binding molecules described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the binding molecules are prepared by introducing appropriate nucleotide changes into the binding molecules nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the binding molecules. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as abolishment of protein A binding and FcγRI binding, or protease-resistance.

Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se needs not to be predetermined. For example, to analyze the performance of a mutation at a given site, Ala scanning or random mutagenesis is conducted at a target codon or region and the expressed binding molecule variants are screened for the desired activity. Preferably, amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

Other modifications of the binding molecule are contemplated herein. For example, the binding molecule may be linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The binding molecule may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th edition, Oslo, A., Ed., (1980), which is hereby incorporated by reference in its entirety.

For therapeutic use, the binding molecules described herein may be prepared as pharmaceutical compositions containing an effective amount of the binding molecules as an active ingredient in a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the agent of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21^(st) Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989, which is hereby incorporated by reference in its entirety.

The binding molecules described herein can be used in non-isolated or isolated form. Furthermore, the binding molecules hereof can be used alone or in a mixture comprising at least one binding molecule hereof. In other words, the binding molecules can be used in combination, e.g., as a pharmaceutical composition comprising two or more binding molecules hereof, variants or fragments thereof. For example, binding molecules having different, but complementary activities can be combined in a single therapy to achieve a desired therapeutic effect, but alternatively, binding molecules having identical activities can also be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect. Optionally, the mixture further comprises at least one other therapeutic agent. In one aspect, the other therapeutic agent may be an anti-infective agent, an antibiotic agent, and/or an antimicrobial agent that is useful in the prophylaxis and/or treatment of a staphylococcal infection. In another aspect, the other therapeutic agent may be an agent that is useful in the prophylaxis and/or treatment of a condition associated with a staphylococcal infection.

A method of producing a binding molecule hereof is an additional part of the disclosure. Such a method comprises the steps of (a) culturing a host cell comprising a vector containing a nucleic acid sequence(s) encoding the binding molecule under conditions suitable for production of the binding molecule, and (b) recovering the binding molecule from the culture. The host cell is preferably a mammalian host cell, such as a non-human mammalian host cell. Accordingly, another aspect relates to nucleic acid molecules encoding the binding molecules (or a fragment thereof). These nucleic acid molecules may be inserted into a vector, i.e., an expression vector, and then transformed (or transfected) in an appropriate host cell and/or expressed in a transgenic animal. The binding molecules (or fragments thereof) so expressed may then be isolated by methods known in the art. See e.g., Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL [3^(rd) Ed.] Cold Spring Harbor Laboratory Press (2001), which is hereby incorporated by reference in its entirety.

The recombinant vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press (1990), which is hereby incorporated by reference in its entirety. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The host cell may be co-transfected with the two or more expression vectors as described herein, the first expression vector containing DNA encoding an operon and a first binding domain (e.g., an antibody or antibody fragment that binds to a glycosylated staphylococcal surface protein) and the second vector containing DNA encoding an operon and a second binding domain (e.g., an alternative scaffold, such as FN3, that binds to a staphylococcal leukotoxin). The two vectors contain different selectable markers, but preferably achieve substantially equal expression of the first binding domain and the second binding domain polypeptides. Alternatively, a single vector may be used, the vector including DNA encoding both the first binding domain and the second binding domain. The coding sequences for the first binding domain and the second binding domain may comprise cDNA, genomic DNA, or both.

The recombinant expression vectors of the invention may be designed for production of binding molecules in prokaryotic or eukaryotic cells. For example, binding molecules of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press (1990), which is hereby incorporated by reference in its entirety. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, to the amino or carboxy terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, PreScission, TEV and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, “Single-Step Purification of Polypeptides Expressed in Escherichia coli as Fusions With Glutathione S-Transferase,” Gene 67:31-40 (1988), which is hereby incorporated by reference in its entirety), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

A recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. For efficient production of the binding molecule described herein, it is preferable to place the nucleotide sequences encoding the binding molecule under the control of expression control sequences optimized for expression in a desired host. For example, the sequences may include optimized transcriptional and/or translational regulatory sequences (e.g., altered Kozak sequences).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (see e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY Academic Press 185:119-128 (1990), which is hereby incorporated by reference in its entirety). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada et al., “Codon Usage Tabulated From the GenBank Genetic Sequence Data,” Nucl. Acids Res. 20:2111-2118 (1992), which is hereby incorporated by reference in its entirety). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques. Another strategy to solve codon bias is by using BL21-codon plus bacterial strains (Invitrogen) or Rosetta bacterial strain (Novagen), these strains contain extra copies of rare E. coli tRNA genes.

The expression vector encoding the binding molecules described herein may be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari et al., “A Novel Leader Peptide Which Allows Efficient Secretion of a Fragment of Human Interleukin 1 beta in Saccharomyces cerevisiae,” EMBO J. 6:229-234 (1987), which is hereby incorporated by reference in its entirety), pMFa (Kurjan and Herskowitz, “Structure of a Yeast Pheromone Gene (MF alpha): a Putative Alpha-Factor Precursor Contains Four Tandem Copies of Mature Alpha-Factor,” Cell 30:933-943 (1982), which is hereby incorporated by reference in its entirety), pJRY88 (Schultz et al., “Expression and Secretion in Yeast of a 400-kDa Envelope Glycoprotein Derived From Epstein-Barr Virus,” Gene 54:113-123 (1987), which is hereby incorporated by reference in its entirety), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.)

Alternatively, the binding molecules described herein can be produced in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., “Production of Human Beta Interferon in Insect Cells Infected With a Baculovirus Expression Vector,” Mol. Cell. Biol. 3:2156-2165 (1993), which is hereby incorporated by reference in its entirety) and the pVL series (Luckow et al., “High Level Expression of Nonfused Foreign Genes With Autographa californica Nuclear Polyhedrosis Virus Expression Vectors,” Virology 170:31-39 (1989), which is hereby incorporated by reference in its entirety). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B., “An LFA-3 cDNA Encodes a Phospholipid-Linked Membrane Protein Homologous to its Receptor CD2,” Nature 329:840 (1987), which is hereby incorporated by reference in its entirety) and pMT2PC (Kaufman et al., “Translational Efficiency of Polycistronic mRNAs and Their Utilization to Express Heterologous Genes in Mammalian Cells,” EMBO J. 6:187-193 (1987), which is hereby incorporated by reference in its entirety), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Sambrook, et al. (Eds.) MOLECULAR CLONING: A LABORATORY MANUAL (3^(rd) Ed.) Cold Spring Harbor Laboratory (2001), which is hereby incorporated by reference in its entirety.

A host cell can be any prokaryotic or eukaryotic cell. For example, binding molecules of the invention can be produced in bacterial cells such as E. coli, insect cells, yeast, plant or mammalian cells (e.g., Chinese hamster ovary cells (CHO), COS, HEK293 cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. [Eds.] MOLECULAR CLONING: A LABORATORY MANUAL (3^(rd) Ed.) Cold Spring Harbor Laboratory and other laboratory manuals (2001), which is hereby incorporated by reference in its entirety.

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. See e.g., Sambrook, et al. (Eds.) MOLECULAR CLONING: A LABORATORY MANUAL (3^(rd) Ed.) Cold Spring Harbor Laboratory (2001) and Walker & Papley MOLECULAR BIOLOGY AND BIOTECHNOLOGY (5^(th) Ed.) Royal Society of Chemistry (2009), which are hereby incorporated by reference in their entirety. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one nucleic acid molecule into a host cell capable of expressing the binding molecule or binding molecules of interest.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the binding molecule or binding molecules as described herein, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) the binding molecule of the invention. Accordingly, the invention further provides methods for producing binding molecules of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector encoding a binding molecule as described herein has been introduced) in a suitable medium such that binding molecule or binding molecules of the invention are produced. In another embodiment, the method further comprises isolating the binding molecules of the invention from the medium or the host cell.

After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the binding molecule or binding molecules of interest, which is then recovered from the culture using standard techniques. Examples of such techniques are well known in the art (see, e.g., WO00/06593 to Zuker et al., which is hereby incorporated by reference in its entirety).

Furthermore, the binding molecules of the present invention can be used for the treatment, prevention or amelioration of a staphylococcal infection. The staphylococcal infection may be caused by any Staphylococcus spp. In one aspect, the staphylococcal infection is caused by Staphylococcus aureus, including MRSA and MSSA strains of S. aureus. Accordingly, the present disclosure provides a method for the treatment, prevention or amelioration of a staphylococcal infection that involves administering to a subject in need thereof a binding molecule as described herein.

In accordance with this aspect, the target “subject” encompasses any animal, for example, a mammal, such as a human. In the context of administering a composition of the invention for purposes of preventing a staphylococcal infection in a subject, the target subject encompasses any subject that is at risk of becoming infected with staphylococcus or developing a staphylococcal infection. Susceptible subjects include infants and juveniles, as well as immunocompromised juvenile, adults, and elderly adults. However, any infant, juvenile, adult, or elderly adult or immunocompromised individual at risk for developing a staphylococcal infection can be treated in accordance with the methods described herein. In the context of administering a composition of the invention for purposes of treating a staphylococcal infection in a subject, the target subject population encompasses any subject infected with staphylococcus. Particularly suitable subjects include those at risk of infection or those infected with methicillin-resistant S. aureus (MRSA) or methicillin sensitive S. aureus (MSSA). Other suitable subjects include those subjects which may have or are at risk for developing a condition resulting from a staphylococcus infection, i.e., a staphylococcal associated condition, such as, for example, skin wounds and infections, tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skin syndrome, septicemia, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.

In one embodiment, the binding molecules described herein are administered prophylactically to prevent, delay, or inhibit the development of staphylococcal infection in a subject at risk of developing a staphylococcal infection or associated condition. In one aspect, prophylactic administration of one or more binding molecules described herein is effective to fully prevent S. aureus infection in an individual. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., substantially prevent, inhibit, or minimize staphylococcal infection in an individual.

In another embodiment, the binding molecules described herein are administered therapeutically to an individual having a staphylococcal infection to inhibit the progression and further development of the infection, i.e., to inhibit and/or prevent the spread of the infection to other cells in an individual, decrease infection, and to treat or alleviate one or more symptoms of infection.

Therapeutically effective amounts of the binding molecules described herein can be determined in accordance with standard procedures, which take numerous factors into account, including, for example, the concentrations of the binding molecules in a pharmaceutical composition, the mode and frequency of administration, the severity of the staphylococcus infection to be treated (or prevented), and subject details, such as age, weight and overall health and immune condition. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990), which is hereby incorporated by reference in its entirety. A clinician may administer a composition comprising the binding molecules described herein in a single dose or in accordance with a multi-dosing protocol until a dosage is reached that provides the desired or required prophylactic or therapeutic effect. The progress of this therapy can be easily monitored by conventional assays. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease.

The therapeutic compositions of the present invention can be administered alone or as part of a combination therapy in conjunction with one or more other active agents, depending upon the nature of the staphylococcus infection that is being treated. Such additional active agents include anti-infective agents, antibiotic agents, and antimicrobial agents that are readily known in the art.

The mode of administration of the binding molecules described herein may be any suitable route that delivers the binding molecule(s) to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal); using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by, for example, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

The binding molecules provided herein can also be used in methods for diagnosing a staphylococcal infection in a subject. In one aspect, the method for diagnosing a staphylococcal infection involves contacting a binding molecule as described herein with a sample from the subject to be diagnosed, and detecting a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample, i.e., detecting the presence or the absence of the glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample. A staphylococcal infection is diagnosed in the subject based on this detection. In other words, the detection of the glycosylated staphylococcal surface protein and/or the staphylococcal leukotoxin indicates a positive diagnosis of a staphylococcal infection.

In accordance with this aspect, the sample from the subject may comprise a blood, tissue, cell, serum, or other biological sample.

Another aspect relates to a method for the detection of a staphylococcal infection in a sample. This method involves contacting the binding molecule as described herein with a sample, and detecting the presence or the absence of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin. The detection of the glycosylated staphylococcal surface protein and/or the staphylococcal leukotoxin indicates the presence of staphylococcus in the sample. In accordance with this aspect, the sample may be any biological sample obtained from the environment, an animal, or a human.

Methods described herein involving the detection of a glycosylated staphylococcal surface protein and/or staphylococcal leukotoxin in a sample from a subject or elsewhere involve the use of a detectably labeled binding molecule. Accordingly, in one aspect the binding molecule as described herein may be coupled to a detectable label. Suitable detectable labels are well known in the art and include detectable tags (e.g., a poly-histidine (His₆₋) tag, a glutathione-S-transferase (GST-) tag, or a maltose-binding protein (MBP-) tag); radioactive labels (e.g., carbon (¹⁴C) or phosphorous (³²P)); fluorescent labels (e.g., fluorescein and derivatives thereof, fluorescein isothiocyanate, rhodamine and derivatives thereof, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); luminescent labels (e.g., luminol); bioluminescent labels (e.g., luciferase, luciferin, and aequorin); or enzymatic labels (e.g., luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase). Alternatively, the binding molecule can be bound by a detectable label, for example, bound by a secondary antibody that contains a detectable label.

Detection assays for detecting the labeled binding molecule bound to a glycosylated staphylococcal surface protein and/or staphylococcal leukotoxin in a sample are well known in the art and include, for example, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescent activated cell sorting (FACS).

Furthermore, the binding molecules of the present invention can be used for the prevention of a staphylococcal infection. This method involves contacting the binding molecule as described herein with a sample from a subject, and detecting a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample as a result of the contacting. If a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin is detected in the subject sample, then an agent suitable for preventing staphylococcal infection is administered to the subject. Exemplary prophylactic agents include, but are not limited to; the binding molecules described herein, one or more antibiotics (e.g., mupirocin, nafcillin, cefazolin, dicloxacillin, clindamycin, vancomycin, linezolid, rifampin, sulfamethoxazole-trimethoprim), and/or other anti-infective agents that are effective against staphylococcal infection.

EXAMPLES

Examples are provided below to illustrate the present invention. These examples are not meant to constrain the present invention to any particular application or theory of operation.

Example 1 Construction and Selection of Fibronectin Type III (FN3) Domains that Bind and Neutralize Leukotoxins

Tencon (SEQ ID NO:1) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain, designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., “Design of Novel FN3 Domains With High Stability by a Consensus Sequence Approach,” Protein Engineering, Design, and Selection 25:107-117 (2012), the disclosure of which is hereby incorporated by reference in its entirety). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands. These loops, or selected residues within each loop, can be randomized in order to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to LukAB, LukD, or LukE

Tencon: (SEQ ID NO: 1) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAINLTVP GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

Procedure

Construction of TCL7 (TCL18) and TCL9 (TCL17) Libraries.

Libraries designed to randomize only the F:G loop of Tencon, TCL9 (TCL17), or a combination of the BC and FG loops, TCL7 (TCL18), were constructed for use with the cis-display system (Odegrip et al., “CIS Display: In Vitro Selection of Peptides From Libraries of Protein-DNA Complexes,” Proc. Natl. Acad. Sci. USA 101:2806-2810 (2004), the disclosure of which is hereby incorporated by reference in its entirety). In this system, linear double-stranded DNA incorporating sequences for a Tac promoter, Tencon library coding sequence, RepA coding sequence, cis-element, and on element is produced. Upon expression in an in vitro transcription/translation system, a complex of the FN3 domain-RepA fusion protein bound in cis to the DNA from which it is encoded is formed. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

DNA fragments encoding the Tencon gene sequence with randomized BC or FG loop sequences were synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were synthesized independently and later combined as described below so that libraries with different combinations of loop lengths were produced. Table 1 below summarizes the design characteristics of these Tencon loop libraries.

TABLE 1 Designed amino acid sequences of the BC and FG loops of the TCL7 (TCL18) and TCL9 (TCL17) libraries. The sequence of the unrandomized Tencon BC and FG loops is shown for com-  parison. X represents a mixture of 18 amino  acids devoid of cysteine and methionine. Library B:C Loop Design F:G Loop Design Tencon TAPDAA KGGHRSN (SEQ ID NO: 3) (SEQ ID NO: 4) TCL7 X₆₋₉ X₇₋₁₀SN (TCL18) TCL9 TAPDAA X₇₋₁₂SN (TCL17) (SEQ ID NO: 3)

Production of FG Loop Libraries (TCL9 (TCL17)).

A set of synthetic DNA molecules consisting of a 5′ Tac promoter followed by the complete gene sequence of Tencon with the exception of randomized codons in the FG loop was produced (U.S. Pat. Publ. No. 2013/0079243 to Diem et al., which is hereby incorporated by reference in its entirety). For FG loop randomization, all amino acids except cysteine and methionine were encoded at equal percentages. The lengths of the diversified portion are such that they encode for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sub-libraries of each length variation were synthesized individually at a scale of 2 ug and then amplified by PCR using oligos Sloning-FOR and Sloning-Rev.

The 3′ fragment of the library is a constant DNA sequence containing elements for display, including a PspOMI restriction site, the coding region of the repA gene, and the cis- and on elements. PCR reactions were performed to amplify this fragment using a plasmid (pCR4Blunt) (Invitrogen) as a template with M13 Forward and M13 Reverse primers. The resulting PCR products were digested by PspOMI overnight and gel-purified. To ligate the 5′ portion of library DNA to the 3′ DNA containing repA gene, 2 pmol (˜540 ng to 560 ng) of 5′ DNA was ligated to an equal molar (˜1.25 ug) of 3′ repA DNA in the presence of NotI and PspOMI enzyme and T4 ligase at 37° C. overnight. The ligated library product was amplified by using 12 cycles of PCR with oligos POP2250 (SEQ ID NO:6) and DigLigRev (SEQ ID NO:7). For each sub-library, the resulting DNA from 12 PCR reactions were combined and purified by Qiagen spin column. The yield for each sub-library of TCL9 (TCL17) ranged from 32-34 ug.

TCL7 (TCL18) Library Construction.

The TCL7 (TCL18) library provides for a library with randomized Tencon BC and FG loops. In this library BC loops of lengths 6-9 amino acids were mixed combinatorially with randomized FG loops of 7-12 amino acids in length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NOs:80-83, respectively) were produced to include the Tencon gene encoding for the N-terminal portion of the protein up to and including residue VX such that the BC loop is replaced with either 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of L17A, N46V and E86I mutations (CEN5243) but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding for randomized FG loops, the following steps were taken.

First, a DNA fragment encoding the Tac promoter and the 5′ sequence of Tencon up to the nucleotide encoding for amino acid A17 (130mer-L17A) (SEQ ID NO:84) was produced by PCR using oligos POP2222ext (SEQ ID NO:85) and LS1114 (SEQ ID NO:86). This was done to include the L17A mutation in the library. Next, DNA fragments encoding for Tencon residues R18-V74 including randomized BC loops were amplified by PCR using BC6, BC7, BC8, or BC9 as a templates and oligos LS1115 (SEQ ID NO:87) and LS1117 (SEQ ID NO:88). This PCR step introduced a BsaI site at the 3′ end. These DNA fragments were subsequently joined by overlapping PCR using oligos POP2222ext and LS1117 as primers. The resulting PCR product of 240 bp was pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified.

Fragments encoding the FG loop were amplified by PCR using FG7, FG8, FG9, FG10, FG11, and FG12 as templates with oligonucleotides SDG10 (SEQ ID NO:89) and SDG24 (SEQ ID NO:90) to incorporate a BsaI restriction site and N46V and E86I variations.

The digested BC fragments and FG fragments were ligated together in the 16 possible combinations as 4 ligation reactions in combinations of two BC loop lengths with 2 FG loop lengths. Each ligation contained ˜300 ng of total BC fragment and 300 ng of the FG fragment. These 4 ligation pools were then amplified by PCR using oligos POP2222 (SEQ ID NO:5) and SDG28 (SEQ ID NO:91). 7.5 ug of each reaction product were then digested with Not1 and cleaned up with a Qiagen PCR purification column. 5.2 ug of this DNA, was ligated to an equal molar amount of RepA DNA fragment (˜14 ug) digested with PspOMI and the product amplified by PCR using oligos POP2222.

Design of TCL19, TCL21, TCL23, and TCL24 Libraries.

The choice of residues to be randomized in a particular library design governs the overall shape of the interaction surface created. X-ray crystallographic analysis of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a largely curved interface that fits into the active site of MBP (Koide et al., “High-Affinity Single-Domain Binding Proteins With a Binary-Code Interface,” Proc. Natl. Acad. Sci. USA 104:6632-6637 (2007), which is hereby incorporated by reference in its entirety. In contrast, an ankyrin repeat scaffold protein that was selected to bind to MBP was found to have a much more planar interaction surface and to bind to the outer surface of MBP distant from the active site (Binz et al., High-Affinity Binders Selected From Designed Ankyrin Repeat Protein Libraries,” Nat. Biotechnol. 22:575-82 (2004), which is hereby incorporated by reference in its entirety). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may dictate what target proteins or specific epitopes on those target proteins are able to be bound effectively by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding have relied on engineering adjacent loop for target binding, thus producing curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops lying on the opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by the beta-strands that form the center of the FN3 structure. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta-strands, the C and the F beta-strands, and is herein called the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. Beta-strands have a repeating structure with the side chain of every other residue exposed to the surface of the protein. Thus, a library can be made by randomizing some or all surface exposed residues in the beta strands. By choosing the appropriate residues in the beta-strands, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interaction with other proteins. A library built upon this premise, called TCL14 was previously described in US2013/0079243A1 to Diem et al, which is hereby incorporated by reference in its entirety. A new library was then created under a very similar design to that of TCL14. Whereas TCL14 used degenerate NNS codons to diversify the Tencon scaffold, TCL19 (SEQ ID NO:86) was generated using DNA fragments encoding the Tencon gene synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Thus, each position shown with an “X” below was replaced with an equal mixture of 18 amino acids (no cysteine or methionine).

TCL19: (SEQ ID NO: 92) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFXIXYXEXXXXGEAIVLTVP GSERSYDLTGLKPGTEYXVXIXGVKGGXXSXPLSAIFTT; wherein “X” is an equal mixture of 18 amino acids (no cysteine or methionine).

TABLE 2 Amino acid SEQ positions (in ID Region  SEQ ID NO: 92) Amino acid sequence NO: C strand 29-37 DSFXIXYXE 93 F strand 65-74 TEYXVXIXGV 94 C strand + CD loop 29-43 DSFXIXYXEXXXXGE 95 F strand + FG loop 65-81 TEYXVXIXGVKGGXXSX 96 A strand + AB loop +  1-28 LPAPKXLXVXXVXXXXAXL 97 B strand + BC loop XWXAPDAAF E strand 55-59 XYXLT 98

Two additional Tencon libraries of similar design were produced. These two libraries, TCL21 and TCL23, are randomized at the same positions as TCL14 (described in US2013/0079243A1 to Diem et al., which is hereby incorporated by reference in its entirety); however, the distribution of amino acids occurring at these positions is altered (Table 3 below). TCL21 was designed to include an equal distribution of 18 natural amino acids at every position (5.55% of each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the CDR-H3 loops of functional antibodies (Birtalan et al., “The Intrinsic Contributions of Tyrosine, Serine, Glycine and Arginine to the Affinity and Specificity of Antibodies,” Journal of Molecular Biology 377:1518-1528 (2008), which is hereby incorporated by reference in its entirety) as described in Table 3. As with the TCL21 library, cysteine and methionine were excluded.

A third additional library was built to expand potential target binding surface of the TCL21 library. In this library, TCL24, 4 additional Tencon positions were randomized as compared to libraries TCL14, TCL21, and TCL23. These positions include N46 and T48 from the D strand and S84 and 186 from the G strand. Positions 46, 48, 84, and 86 were chosen in particular as the side chains of these residues are surface exposed from beta-strands D and G and lie structurally adjacent to the randomized portions of the C and F strand, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL21 and in Table 3.

TCL24: (SEQ ID NO: 667) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFXIXYXEXXXXGEAIXLXVP GSERSYDLTGLKPGTEYXVXIXGVKGGXXSXPLXAXFTT wherein “X” represents an equal mixture of 18 natural amino acids.

TABLE 3 Amino acid frequency (%) at each randomized position for TCL21, TCL23, and TCL24. Amino Acid TCL21 TCL23 TCL24 Ala 5.6 6.0 5.6 Arg 5.6 6.0 5.6 Asn 5.6 3.9 5.6 Asp 5.6 7.5 5.6 Cys 0.0 0.0 0.0 Gln 5.6 1.5 5.6 Glu 5.6 2.5 5.6 Gly 5.6 15.0 5.6 His 5.6 2.3 5.6 Ile 5.6 2.5 5.6 Leu 5.6 5.0 5.6 Lys 5.6 1.5 5.6 Met 0.0 0.0 0.0 Phe 5.6 2.5 5.6 Pro 5.6 4.0 5.6 Ser 5.6 10.0 5.6 Thr 5.6 4.5 5.6 Trp 5.6 4.0 5.6 Tyr 5.6 17.3 5.6 Val 5.6 4.0 5.6

Generation of TCL21, TCL23, and TCL24 libraries. The TCL21 library was generated using Colibra library technology (Isogenica) in order to control amino acid distributions. TCL23 and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distributions. PCR was used to amplify each library following initial synthesis followed by ligation to the gene for RepA in order to be used in selections using the CIS-display system as previously described (Odegrip et al., “CIS Display: In Vitro Selection of Peptides From Libraries of Protein-DNA Complexes,” Proc. Natl. Acad. Sci. USA 101:2806-2810 (2004), which is hereby incorporated by reference in its entirety).

Library Screening.

Cis-display was used to select Leukotoxin binding domains from the TCL17, TCL18 and TCL19 libraries. Recombinant LukA′B toxoid complex (SEQ ID NOs:10 and 11), LukD (SEQ ID NO:12), and LukE (SEQ ID NO:13) leukotoxins were biotinylated using standard methods and used for panning Although the modified LukA′B toxoid complex was used for panning in the present Example, binding and/or neutralization of the unmodified LukAB toxin has been confirmed. For in vitro transcription and translation (ITT), 2-6 μg of library DNA were incubated with 0.1 mM complete amino acids, 1λ S30 premix components, and 30 μL of S30 extract (Promega) in a total volume of 100 μL and incubated at 30° C. After 1 hour, 450 μL of blocking solution (PBS pH 7.4, supplemented with 2% bovine serum albumin, 100 μg/mL herring sperm DNA, and 1 mg/mL heparin) were added and the reaction was incubated on ice for 15 minutes. For binding, 500 μL of blocked ITT reactions were mixed with 100 μL of biotinylated leukotoxins and incubated for 1 hour at room temperature, after which bound complexes were pulled down with magnetic neutravidin or streptavidin beads (Promega and Invitrogen). Unbound library members were removed by successive washes with PBST and PBS. After washing in rounds five, seven, and nine, DNA was eluted from the bound complexes by heating to 65° C. for 10 minutes, amplified by PCR, and attached to a DNA fragment encoding RepA by restriction digestion and ligation for further rounds of panning (this step is optional when repA is incorporated into the library). High affinity binders were isolated by successively lowering the concentration of the target leukotoxin during each round from 400 nM to 2.5 nM and increasing the washing stringency. Panning was performed through seven (LukE) and nine (LukAB and LukD) successive rounds.

Following panning, selected FN3 domains were amplified by PCR using oligos MDD40 (SEQ ID NO:8) and MDD62 (SEQ ID NO:9), subcloned into a pET vector modified to include a ligase independent cloning site, and transformed into BL21-GOLD (DE3) (Stratagene) cells for soluble expression in E. coli using standard molecular biology techniques. A gene sequence encoding a C-terminal poly-histidine tag was added to each FN3 domain to enable purification and detection. Cultures were grown to an optical density of 0.6-0.8 in 2YT medium supplemented with 100 μg/mL carbenicillin in 1 mL 96-well blocks at 37° C. before the addition of IPTG to 1 mM, at which point the temperature was reduced to 30° C. Cells were harvested approximately 16 hours later by centrifugation and frozen at −20° C. Cell lysis was achieved by incubating each pellet in 0.6 mL of BugBuster® HT lysis buffer (Novagen EMD Biosciences) with shaking at room temperature for 45 minutes. Antigen binding of nickel-purified FN3 domains is confirmed by ELISA.

Results

FN3 domains which bound to LukA′B (toxoid), LukD, and LukE were identified by ELISA following cis-display panning of each toxin/toxoid molecule. DNA sequences of binders were identified and unique sequences were isolated. Monomeric proteins expressed from these sequences were identified and expected molecular weights were confirmed. Antigen binding, sequence identification numbers, and monomeric FN3 domains are reported in Table 4.

TABLE 4 Characterization of FN3 domains that bind to LukAB, LukD and LukE. SEQ ID NO: Description Diversity Regions Target 14 Luk17 C, CD, F, FG LukA′B 15 Luk19 BC, FG LukA′B 16 Luk20 BC, FG LukA′B 17 Luk24 C, CD, F, FG LukA′B 18 Luk8 BC, FG LukD 19 Luk9 BC, FG LukD 20 Luk10 BC, FG LukD 21 Luk11 BC, FG LukD 22 Luk12 BC, FG LukD 23 Luk21 BC, FG LukD 24 Luk22 BC, FG LukD 25 Luk26 C, CD, F, FG LukE 26 Luk27 C, CD, F, FG LukE 27 Luk28 C, CD, F, FG LukE 28 Luk29 BC, FG LukE 29 Luk30 BC, FG LukE 30 Luk31 BC, FG LukE 31 Luk32 BC, FG LukE 32 Luk33 FG LukE 33 Luk34 FG LukE 34 Luk35 FG LukE 35 Luk36 FG LukE 36 Luk37 FG LukE 37 Luk38 FG LukE 38 Luk39 FG LukE 39 Luk40 FG LukE 40 Luk41 FG LukE 41 Luk42 FG LukE 42 Luk43 FG LukE 43 Luk44 FG LukE 44 Luk45 FG LukE 45 Luk46 C, CD, F, FG LukE 46 Luk47 C, CD, F, FG LukE 47 Luk48 C, CD, F, FG LukE 48 Luk49 C, CD, F, FG LukE 49 Luk50 C, CD, F, FG LukE 50 Luk51 C, CD, F, FG LukE 51 Luk52 C, CD, F, FG LukE 52 Luk53 C, CD, F, FG LukE 53 Luk54 C, CD, F, FG LukE 54 Luk55 C, CD, F, FG LukE 55 Luk56 C, CD, F, FG LukE 56 Luk57 C, CD, F, FG LukE 57 Luk58 C, CD, F, FG LukE 58 Luk59 C, CD, F, FG LukE 59 Luk60 C, CD, F, FG LukE

Additional FN3 domains the bind LukAB, LukD, and LukE are disclosed infra as SEQ ID NOs:113-666 in Table 6.

Summary

Cis-display panning successfully identified sequence unique, monomeric, staph leukotoxin binding FN3 domains targeted to LukA′B (4), LukD (7), and LukE (35) as detected by ELISA.

Example 2 Neutralization of Leukotoxin-Mediated Immune Cell Killing by Anti-LukD FN3 Domains

To evaluate the functionality of the isolated anti-LukD FN3 domains, a cell-based cytotoxic neutralization assay was developed. Three types of FN3 domains were used, FN3 domains without linkers, and FN3 domains with the GS or the GSM linker.

Procedure

To screen the anti-LukD FN3 domains for neutralizing activity, 75 nM of LukED; a dose required to kill 100% of the cells or LD₁₀₀; was incubated for 30 minutes on ice with increasing concentrations of the different FN3 domains in a final volume of 30 μl. Freshly isolated primary human neutrophils (hPMNs), 2×10⁵ cells in 70 μl of RPMI+10 mM HEPES+0.1% HAS, were then added to the toxin-FN3 domain mixtures. Following 1 hr incubation in a 37° C. 5% CO₂ incubator, 10 μl of the Cell Titer AQ ONE Solution (Promega), was added to the cells and incubated for 1 hour. Cell Titer is a colorimetric assay that evaluates cell viability by monitoring cellular metabolic activity. The colorimetric reaction was measured at 492 nm using a Perkin Elmer Envision Multilabel Reader. Percent viable cells were calculated using the following equation: % Viability=100×[(Ab₄₉₂Sample−Ab₄₉₂TritonX100)/(Ab₄₉₂Tissue culture media)].

Membrane damage was also measured by monitoring lactate dehydrogenase (LDH) release one hour post-incubation. 50 μl of supernatant was removed and added to wells containing 50 μl of LDH reagent (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega) and incubated for an additional 30 min at room temperature. LDH activity was measured using a Perkin Elmer Envision Multilabel Reader (Excitation 555 nm, Emission 590 nm) and data were normalized to 100% PMN lysis induced by 0.1% v/v Triton-X100.

Results

As shown in FIG. 1, the ability of selected anti-LukD FN3 domains to neutralize the cytotoxic activity of LukED towards human PMNs was assessed. Three selected anti-LukD FN3 domains; Luk10, Luk12, and Luk22; were found to block LukED-mediated killing of hPMNs. This inhibition was observed when measuring cell metabolism (Cell Titer) as well as membrane damage (LDH). In contrast to the anti-LukD FN3 domains, no neutralization was observed with the TENCON control FN3 domain.

Summary

As expected, the TENCON molecule was unable to block the cytotoxic potential of LukED In contrast, the selected anti-LukD FN3 domains neutralized LukED irrespectively of whether or not they were fused to a linker. The anti-LukD FN3 domains blocked LukED-mediated cytotoxicity in a dose-dependent manner. For illustrative purposes, the Luk12 anti-LukD FN3 domains were chosen for the generation of mAb-FN3 domain conjugates.

Example 3 Neutralization of Leukotoxin-Mediated Immune Cell Killing by Anti-LukAB FN3 Domains

To evaluate the functionality of the isolated anti-LukA′B FN3 domains, a cell-based cytotoxic neutralization assay was developed and employed. Three types of FN3 domains were used, FN3 domains without linkers, and FN3 domains with the GS or the GSM linker.

Procedure

To screen the anti LukA′B FN3 domains for neutralizing activity, 18.75 nM of the active LukAB toxin (LD₁₀₀) was incubated for 30 minutes on ice with increasing concentrations of different versions of FN3 domains Luk17 (+/−linkers) in a 96-well black well clear bottom tissue culture-treated plate. 2×10⁵ freshly isolated primary hPMNs in phenol red-free RPMI+10 mM HEPES+0.1% HSA were then added to the toxin-FN3 domain complexes and samples were incubated for one hour in a 37° C. 5% CO₂ incubator. Membrane damage was evaluated by two assays: ethidium bromide permeability and lactate dehydrogenase (LDH) release. Ethidium bromide permeability was measured using a Perkin Elmer Envision Multilabel Reader (Excitation 530 nm, Emission 590 nm). LDH release into the culture supernatant was also measured one hour post-incubation. 50 μl of supernatant was removed and added to wells containing 50 μl of LDH reagent (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega) and incubated for an additional 30 minutes at room temperature. LDH activity was measured using a Perkin Elmer Envision Multilabel Reader (Excitation 555 nm, Emission 590 nm) and data were normalized to 100% PMN lysis induced by 0.1% v/v Triton-X100.

Results

As shown in FIG. 2, the ability of the Luk17 anti-LukAB FN3 domains to neutralize the cytotoxic activity of LukAB towards human PMNs was assessed. The anti-LukAB FN3 domains were found to block LukAB-mediated killing of hPMNs as evaluated by ethidium permeability and LDH release. In contrast, no neutralization was observed with the control FN3 domain molecules.

Summary

As expected, the TENCON molecule was unable to block the cytotoxic potential of LukAB. In contrast, the anti-LukAB FN3 domains neutralized LukAB cytotoxicity irrespectively of whether or not they were fused to a linker. The anti-LukAB FN3 domains blocked LukAB-mediated cytotoxicity in a dose dependent manner. For illustrative purposes, the Luk17 anti-LukAB FN3 domain was chosen for the generation of the mAB-FN3 domain conjugates.

Example 4 Neutralization of Leukotoxin-Mediated Immune Cell Killing by Anti-LukE FN3 Domains

To evaluate the functionality of the isolated anti-LukE FN3 domains, the same cell-based cytotoxic neutralization assay described for evaluation of anti-LukD FN3 domain was employed. For illustrative purposes, five anti-LukE FN3 domains were used (FN3 domains without linkers).

Procedure

To screen the anti-LukE FN3 domains for neutralizing activity, 75 nM of LukED; a dose required to kill 100% of the cells or LD₁₀₀; was incubated for 30 minutes on ice with increasing concentrations of the different FN3 domains in a final volume of 30 μl. Freshly isolated primary hPMNs, 2×10⁵ cells in 70 μl of RPMI+10 mM HEPES+0.1% HAS, were then added to the toxin-FN3 domain mixtures. Following 1 hr incubation in a 37° C. 5% CO₂ incubator, cell membrane destabilization was monitored via lactate dehydrogenase (LDH) release. LDH release into the culture supernatant was measured one hour post-incubation. 50 μl of supernatant was removed and added to wells containing 50 μl of LDH reagent (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega) and incubated for an additional 30 min at room temperature. LDH activity was measured using a Perkin Elmer Envision Multilabel Reader (Excitation 555 nm, Emission 590 nm) and data were normalized to 100% PMN lysis induced by 0.1% v/v Triton-X100.

Results

The ability of selected anti-LukE FN3 domains to neutralize the cytotoxic activity of LukED towards human PMNs was assessed. All the tested anti-LukE FN3 domains were found to block LukED-mediated killing of hPMNs (FIG. 3).

Summary

LukED neutralization by the anti-LukE FN3 domains was dose dependent. Among the tested FN3 domains, anti-LukE FN3 domains Luk26, Luk27, and Luk38 exhibited the highest neutralizing activity.

Example 5 Construction of Fusion Proteins

Constructs were designed utilizing mAb 5133 (SEQ ID NOs:60 and 61), described in U.S. Pat. No. 8,460,666 to Throsby et al., which is hereby incorporated by reference in its entirety, with the intent to engage a Staph antigen via the Fab V-regions and have leukotoxin-neutralizing constructs linked to either the C-terminus of the light chain or the C-terminus of the heavy chain. Table 5 below summarizes the fusion protein constructs designed.

The left construct of FIG. 4A depicts a monospecific, bivalent mAb with a LukAB neutralizing construct attached to the C-terminus of each heavy chain. The right construct of FIG. 4A depicts a monospecific, bivalent mAb with a LukD or LukE neutralizing construct attached to the C-terminus of each heavy chain.

The left construct of FIG. 4B depicts a monospecific, bivalent mAb with a LukAB neutralizing construct attached to the C-terminus of each light chain. The right construct of FIG. 4B depicts a monospecific, bivalent mAb with a LukD or LukE neutralizing construct attached to the C-terminus of each light chain.

The left construct of FIG. 4C depicts a monospecific, bivalent mAb with a LukAB neutralizing construct attached to the C-terminus of each light chain, and a LukD or LukE neutralizing construct attached to the C-terminus of each heavy chain. The right construct of FIG. 4C depicts a monospecific, bivalent mAb with a LukD or LukE neutralizing construct attached to the C-terminus of each light chain and a LukAB neutralizing construct attached to the C-terminus of each heavy chain.

The left construct of FIG. 4D depicts a monospecific, bivalent mAb with a tandem LukAB neutralizing construct attached to the C-terminus of each heavy chain and a LukD or LukE neutralizing construct attached to the C-terminus of the LukAB neutralizing constructs. The right construct of FIG. 4D depicts a monospecific, bivalent mAb with a tandem LukD or LukE neutralizing construct attached to the C-terminus of each heavy chain and a LukAB neutralizing construct attached to the C-terminus of the LukD or LukE neutralizing constructs.

FIG. 4E depicts a bispecific, bivalent mAb with a LukD or LukE neutralizing construct attached to the C-terminus of the heavy chain recognizing Staph antigen 2, and a LukAB neutralizing construct attached to the C-terminus of the heavy chain recognizing Staph antigen 1.

FIG. 4F depicts a monospecific, bivalent mAb with a LukAB neutralizing construct attached to the C-terminus of one heavy chain, and a LukD or LukE neutralizing construct attached to the C-terminus of the opposing heavy chain.

TABLE 5 Design of fusion protein constructs. Heavy Light Con- SEQ Chain Chain struct ID Descrip- Heavy FN3 FN3 No. NO: tion V-region Chain domains domains 1 60 HC CR5133 anti- IgG1 wt none none 61 LC glycosylated SDR- containing protein 2 62 HC CR5133 anti- E223P/ none none 63 LC PRASA glycosylated L234V/ SDR- L235A containing (G236- protein deleted) S239D/ K326A/ E333A 3 64 HC CR5133 A6 anti- H435R/ none none 65 LC glycosylated Y436F SDR- containing protein 4 66 HC CR5133 anti- E223P/ none none 67 LC PRASA A6 glycosylated L234V/ SDR- L235A containing (G236- protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 5 68 HC CR5133 anti- E223P/ none anti- 69 LC PRASA A6 glycosylated L234V/ LukD LC-D SDR- L235A containing (G236- protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 6 70 HC CR5133 anti- E223P/ anti- none 71 LC PRASA A6 glycosylated L234V/ LukAB HC-AB SDR- L235A containing (G236- protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 7 72 HC CR5133 anti- E223P/ anti- anti- 73 LC PRASA A6 glycosylated L234V/ LukAB LukD LC-D SDR- L235A HC-AB containing (G236- protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 8 74 HC CR5133 anti- E223P/ anti- none 75 LC PRASA A6 glycosylated L234V/ LukD HC-D SDR- L235A containing (G236- protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 9 76 HC CR5133 anti- E223P/ anti- none 77 LC PRASA A6 glycosylated L234V/ LukAB HC AB-D SDR- L235A anti- containing (G236- LukD protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F 10 78 HC CR5133 anti- E223P/ anti- none 79 LC PRASA A6 glycosylated L234V/ LukD HC D-AB SDR- L235A anti- containing (G236- LukAB protein deleted) S239D/ K326A/ E333A/ H435R/ Y436F

Example 6 MAb 5133 Binds Strongly to Different Strains of S. aureus but does not Bind to a sdgB Mutant Strain

The mAb 5133 was previously shown to bind to different strains of S. aureus and possess opsonophagocytic activity (described in U.S. Pat. No. 8,460,666 to Throsby et al., which is hereby incorporated by reference in its entirety). In order to further assess the specificity of the mAb 5133, western blot binding experiments were performed using different strains of S. aureus, as well as sub-strains from the USA300 parental JE2 strain where specific genes have been disrupted using sequence defined transposon mutants (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes,” mBio 4(1):e00537-12 (2013), which is hereby incorporated by reference in its entirety) obtained from the Network on Antibiotic Resistance in Staphylococcus aureus (NARSA).

Procedure

For the preparation of cell lysates, the method used followed that of Brady et al., “Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection,” Infect. Immun. 74:3415-3426 (2006), which is hereby incorporated by reference in its entirety. Staphylococcus aureus strains were cultured in tryptic soy broth at 37° C., 190 rpm overnight. For strains carrying a transposon insertion, 10 μg/mL erythromycin was included in the culture medium. Cultures were diluted 1:100 into fresh medium and cultured for 5-6 hours, to an OD₆₀₀ of approximately 1.2. For each strain, 4.5 mL of culture were centrifuged, and the cell pellets resuspended in 75 μL of lysis buffer (50 mM Tris-HCl, 20 mM MgCl₂ and 10 μg/mL lysostaphin). Lysis was complete following a 20 minute incubation at 37° C. Lysates were then centrifuged at 6,000 rpm, 4° C. for 20 minutes and supernatants transferred to a new tube. For Western blot analysis, 12 μL of each lysate were heated at 70° C. for 10 minutes (LDS sample buffer with 50 mM DTT) and electrophoresed on a 10% Bis-Tris gel (Novex NuPage, Life Technologies) in MOPS buffer at 150 volts for 2 hours. The gel was blotted onto PVDF membrane using the iBlot instrument (Life Technologies) and probed with CR5133 using the FemtoMax kit (Rockland).

Results

As shown in FIG. 5, mAb 5133 detected high molecular weight bands (i.e., above 50 kDa) in lysates generated from S. aureus strain ATCC 29213 and the parental USA300 strain JE2. High molecular weight bands were also detected with NARSA strains deficient in the serine-aspartate repeat (SDR) containing proteins SdrC, SdrD, and SdrE (lane 3 NE432 (sdrC mutant), lane 4 NE1289 (sdrD mutant), lane 5 NE98 (sdrE mutant). Faint bands were detected in the sortase mutant (lane 8 NE1787 (srtA mutant) and a protein A mutant (lane 9 NE286 (protein A mutant). High molecular weight bands were not detected in the glycosyltransferase sdgB mutant (lane 7 NE105 (sdgB mutant)) but were detected in the glycosyltransferase sdgA mutant (lane 6 NE381 (sdgA mutant)).

Summary

Serines within the SDR domain are modified by the sequential addition of N-acetylglucosamines (GlcNAc), first by SdgB followed by SdgA (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog. 9(10):e1003653 (2013), which is hereby incorporated by reference in its entirety). The loss of detection of high molecular weight bands with mAb 5133 in lysates from the sdgB mutant is consistent with 5133 recognizing an SDR epitope that has been glycosylated by SdgB.

Example 7 V8 Proteolytic Digestion of Fusion Proteins

Human IgG1 is susceptible to cleavage in the lower hinge region, and this cleavage can result in a loss of Fc mediated effector function both in vitro and in vivo (Brerski et al., “Tumor-Associated and Microbial Proteases Compromise Host IgG Effector Functions by a Single Cleavage Proximal to the Hinge,” PNAS 106:17864-17869 (2009), which is hereby incorporated by reference in its entirety. The S. aureus protease, GluV8, cleaves human IgG1 in the lower hinge region between amino acids E233 and L234, and it was previously demonstrated that this cleavage abrogates both ADCC and CDC function (Brerski et al., “Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs,” J. Immunol. 181:3183-3192 (2008), which is hereby incorporated by reference in its entirety). In order for the mAb/FN3 domains constructs to function effectively, they must be able to both bind to a S. aureus-associated antigen and engage immune cell FcγRs or complement in order to facilitate phagocytosis. Proteolytic cleavage could uncouple the ability of the mAb to facilitate phagocytosis by immune effector cells. Therefore, the hinge region was engineered to have increased resistance to proteolysis by GluV8.

Procedure

The S. aureus protease GluV8 (cat. E-006) was obtained from BioCentrum. Antibody concentrations were fixed at 3.3 μM in PBS and reactions were initiated by the addition of GluV8 at 0.66 μM to give a 20% molar ratio for GluV8. Digestions were performed at pH 7.5 at 37° C. for 24 hours. Samples were prepared under denaturing conditions and analyzed using the Agilent 2100 microfluidics-based Bioanalyzer. The percent intact IgG was calculated based on the electropherogram profiles generated by capillary electrophoresis (Agilent Technologies) by dividing the percent intact IgG remaining in the protease digested sample by the percent intact IgG remaining in the control sample without protease. Data were analyzed and plotted using GraphPad Prism.

Results

As shown in FIG. 6, constructs IgG1 (IgG1 isotype control) and A6 (construct 3 of Table 5), which contain the lower hinge of human IgG1 wt (E233/L234/L235/G236) displayed a range of 15-25% intact IgG remaining after a 24 hour digest with GluV8. Constructs PRASA A6 (construct 4 of Table 5) and PRASA A6 HC-AB (construct 6 of Table 5), which had a mutated lower hinge (E233P/L234V/L235A with G236 deleted) displayed increased resistance to GluV8 with greater than 70% intact IgG remaining after the 24 hour GluV8 digestion.

Summary

These results indicated that mutation of the lower hinge region of IgG1 conferred increased resistance to GluV8 digestion. Further, the inclusion of the anti-LukAB FN3 domains on the C-terminus of the heavy chain did not affect susceptibility to GluV8 cleavage, because the total amount of intact IgG for the lower hinge mutated construct without the FN3 domains, PRASA A6 (construct 4), had a similar percent intact IgG remaining as the lower hinge mutated construct containing the FN3 domains, PRASA A6 HC-AB (construct 6).

Example 8 Fey Receptor Binding Measurements Using AlphaScreen® Competition Binding Assays

Competition ALPHASCREEN® assays were employed in order to assess the ability of anti-Staph targeting constructs to bind to FcγRs. The constructs were originally designed to bind to human FcγRs, so the constructs were tested for their ability to bind to the human activating FcγRs: FcγRI, FcγRIIa (R131), and FcγRIIIa (V158), and the inhibitory receptor, FcγRIIb.

Procedure

Competition binding studies were carried out in half-well volume 96-well opaque plates (Corning) in assay buffer (PBS, 0.05% BSA, 0.01% Tween-20) at pH 7.4. All competition studies were carried out against biotinylated IgG1 wild type (1 IgG: 2 biotin, using EZ Link™ NHS-LC-biotin, Pierce) at a fixed concentration and with competing IgG1 wild type and anti-Staph constructs in serial 3-fold dilutions. The final concentration of FcγR in the assays was 0.2 μg/ml. Biotinylated IgG1 (0.2 μg/ml final) and IgG1 wild type and anti-Staph constructs (10 μl) were sequentially added to each row of a 96-well plate in duplicates. Thereafter, the designated FcγRs were added followed by the sequential addition of 10 μl each of 1/50 diluted nickel chelate (Ni)-acceptor beads and 1/50 diluted streptavidin (SA)-donor beads. The opaque plate was covered with an aluminum seal to maintain light-safe conditions while shaking for 30 minutes on an Orbital shaker. Thereafter, the seal was removed and the fluorescence was read on an ENVISION™ plate reader (PerkinElmer) equipped with appropriate filter set of AlphaScreen® excitation/emission spectra. Raw data was transferred to GraphPad PRISM™ software and normalized for maximal signal and competition curves were plotted using non-linear regression curve-fitting software. The competition binding curves were generated using the following equation: % Maximal Signal=(Exp−Min)/(Max−Min)×100% where the minimum (Min) was determined by signal detected in the presence of the highest concentration of test article mAb and the maximum (Max) was determined by the signal detected in wells that only contained the biotinylated IgG1, FcγR, nickel chelate (Ni)-acceptor beads and streptavidin (SA)-donor beads without any of the anti-Staph constructs.

Results

The ability of several of the constructs to compete for binding to the human Fc gamma family of receptors with human IgG1 wild type was assessed. The ability of the constructs to reduce maximum signal produced by the biotinylated IgG1 wild type are shown in FIGS. 7A through 7D. As shown in FIG. 7A, the IgG1 (IgG1 isotype control) and A6 (construct 3) bound robustly to human FcγRI. The constructs PRASA A6 (construct 4), PRASA A6 LC-D (construct 5), PRASA A6 HC-AB (construct 6), and PRASA A6 LC-D HC-AB (construct 7) did not display binding to human FcγRI at any of the concentration tested. The constructs IgG1 (IgG1 isotype control), A6 (construct 3), PRASA A6 (construct 4), PRASA A6 LC-D (construct 5), PRASA A6 HC-AB (construct 6), and PRASA A6 LC-D HC-AB (construct 7) all displayed comparable binding to human FcγRIIa (R131) (FIG. 7B). The constructs IgG1 (IgG1 isotype control) and A6 (construct 3) displayed the highest binding to the inhibitory human FcγRIIb, whereas the constructs PRASA A6 (construct 4), PRASA A6 LC-D (construct 5), PRASA A6 HC-AB (construct 6), and PRASA A6 LC-D HC-AB (construct 7) displayed low to undetectable binding (FIG. 7C). The constructs IgG1 (IgG1 isotype control), A6 (construct 3), PRASA A6 (construct 4), PRASA A6 LC-D (construct 5), PRASA A6 HC-AB (construct 6), and PRASA A6 LC-D HC-AB (construct 7) all displayed comparable binding to human FcγRIIIa (V158) (FIG. 7D).

Summary

None of the constructs with the lower hinge mutations (E233P, L234V, L235A with G236-deleted) displayed binding to human FcγRI (see US Patent Publ. No. 2013/0011386 A1), and the inclusion of either a C-terminal FN3 domains on either the heavy or light chain did not affect binding. All constructs displayed comparable binding to activating human FcγRIIa (R131) and FcγRIIIa (V158), which indicated that the inclusion of the FN3 domains did not affect binding to these receptors. The constructs PRASA A6 (4), PRASA A6 LC-D (5), PRASA A6 HC-AB (6), and PRASA A6 LC-D HC-AB (7) displayed low to undetectable binding to the inhibitory receptor FcγRIIb. This could be advantageous since the low binding to human FcγRIIb and normal binding to the activating receptors FcγRIIa and FcγRIIIa would increase the activating (A) to inhibitory (I) index, a property that is thought to increase the efficacy of mAbs with an intended cytotoxic mechanism of action (Nimmerjahn and Ravetch, “Divergent Immunoglobulin g Subclass Activity Through Selective Fc Receptor Binding,” Science 310:1510-1512 (2005), which is hereby incorporated by reference in its entirety).

Example 9 Protein A Binding Measurements

Plate-based ELISA assays were employed in order to assess the binding of the constructs to Protein A coated onto an ELISA plate. Protein A is a Staph virulence factor, and human IgG1 wildtype antibodies are sequestered on the surface of Staph via binding to the Fc of IgG1 wildtype. This process is thought to abrogate the ability of anti-Staph mAbs to bind to surface determinants through the antibody V-region and simultaneously block Fc interactions with humoral and cellular components of the host immune system. In order to circumvent this Staph virulence factor, constructs were engineered with the intent to remove Protein A binding.

Procedure

Direct Protein A binding ELISAs were carried out in clear 96-well plates (Nunc) in ELISA blocking buffer (3% Bovine Serum Albumin in PBS). Biotinylated Protein A was coated in the 96-well plates at 2 μg/ml in PBS for 1 hour at room temperature, washed 3 times in ELISA wash buffer (0.15 M NaCl with 0.02% Tween-20). Plates were then blocked with ELISA blocking buffer for 1 hour at room temperature. Plates were then washed 3 times in ELISA wash buffer. Constructs were applied to the plate with a starting concentration of 10 μg/ml, then were serially diluted 3-fold across the plate and incubated for 1 hour at room temperature. Plates were washed 3 times with ELISA wash buffer. Constructs were detected with a horse radish peroxidase (HRP) conjugated goat anti-human kappa light chain antibody (Millipore) diluted 1:15,000 in ELISA blocking buffer and samples were incubated for 30 minutes at room temperature. Plates were washed 5 times with ELISA wash buffer. The substrate for HRP in this assay was TMB (Fitzgerald), and plates were incubated until to wells turned blue. The reaction was stopped by adding and equal volume of 0.5 M HCl to the TMB, and plates were read at 450 nm. Data were analyzed with Graph Pad Prism software using non-linear regression curve-fits.

Results

The human IgG1 wild type construct (construct 1) displayed detectable binding to Protein A. All of the constructs which contained the CH3 mutation H435R/Y436F (i.e., A6 (construct 3), PRASA A6 (construct 4), PRASA A6 LC-D (construct 5), PRASA A6 HC-AB (construct 6), and PRASA A6 LC-D HC-AB (construct 7) did not display detectable binding to Protein A at any of the concentrations tested (FIG. 8).

Summary

The CH3 mutations H435R/Y436F successfully eliminated Protein A binding, as assessed in this assay. The inclusion of FN3 domains on either the C-terminus of the heavy chain or the light chain did not affect Protein A binding.

Example 10 Binding to Whole Bacteria as Measured by Flow Cytometry

Binding of mAb and FN3 domain-mAb conjugates to the surface of the Newman Strain of S. aureus was assessed using flow cytometric analysis. Because the S. aureus virulence factor Protein A can bind to IgG1 via the Fc portion of the antibody and can confound analysis, antibodies with the H435R/Y436F mutations, designated A6, were used to eliminate non-specific binding of the Fc region of the mAbs to Protein A expressed on the surface of the Staph (see Example 9). Therefore, A6 (construct 3), PRASA A6 (construct 4) and mAb-FN3 domain conjugates each containing the H435R/Y436F mutations were compared. A construct with V-regions specific for lipoteichoic acid (LTA) containing the constant region mutations of E233P/L234V/L235A (G236-deleted) H435R/Y436F (termed “PRASA A6 isotype control”) was used as a negative control as the binding of this construct was low/negative for the S. aureus strain selected.

Procedure

The S. aureus Newman strain was grown at 37° C. with shaking at 225 rpm in brain heart infusion (BHI) modified broth until the cultures reached an OD 600 nM of approximately 0.5. Aliquots were frozen in cryovials with 10% glycerol (final). A vial was thawed and used to determine colony forming units by serial dilution and growth on BHI agar plates. For flow cytometric analysis, Newman strain was plated at 5×10⁶ colony forming units per well and incubated with the indicated serial dilutions of mAb 5133 constructs for 30 minutes at room temperature in FACS buffer. Samples were washed twice with FACS buffer, prior to staining with 20 μg/ml Alexa647 conjugated F(ab′)₂ goat anti-human IgG (heavy and light) to detect the constructs. Samples were analyzed using a BD Fortessa and Flowjo software. These experiments were repeated twice with similar results.

Results

As shown in FIG. 9, as assessed by flow cytometry binding, the constructs A6 (construct 3), PRASA A6 (construct 4), and PRASA A6 HC-AB (construct 6) had similar binding to the Newman S. aureus strain. Addition of an anti-LukD FN3 domain to the light chain resulted in an approximately 2 to 3-fold decrease in binding for constructs PRASA A6 LC-D (construct 5) and PRASA A6 LC-D HC-AB (construct 7) compared to than the same antibody without FN3 domain conjugates (calculated by EC50 test mAb/EC50 PRASA A6 (construct 4)).

Summary

Addition of anti-LukAB to the heavy chain did not impact antibody binding to S. aureus, whereas the addition of the anti-LukD FN3 domain to the C-terminus of the light chain resulted in slightly decreased detection of binding to the S. aureus Newman strain (2 to 3-fold change in EC50 in two independent experiments).

Example 11 Neutralization of Leukotoxin-Mediated Immune Cell Killing by mAb-FN3 Domain Conjugates

To evaluate the functionality of the mAb-FN3 domain 5133 conjugates, the cell-based cytotoxic neutralization assay described for evaluation of neutralization of LukED and LukAB were employed.

Procedure

To screen the mAb-FN3 domain 5133 conjugates for LukED and LukAB neutralizing activity, 75 nM of LukED and 18.75 nM of active LukAB toxin were independently incubated for 30 minutes on ice with increasing concentrations of the different mAb variants (IgG1 isotype control; A6 construct 3; PRASA A6 construct 4); PRASA A6 LC-D construct 5; PRASA A6 HC-D construct 8; PRASA A6 HC-AB construct 6; PRASA A6 LC-D HC-AB construct 7; PRASA A6 HC AB-D construct 9; and PRASA A6 HC D-AB construct 10) in a final volume of 30 μl. Freshly isolated primary human neutrophils (hPMNs); 2×10⁵ cells in 70 μl of RPMI+10 mM HEPES+0.1% HAS were then added to the toxin-mAb mixtures and incubated for 1 hour in a 37° C. 5% CO₂ incubator. Membrane damage was measured by monitoring lactate dehydrogenase (LDH) release one hour post-incubation. 50 μl of supernatant was removed and added to wells containing 50 μl of LDH reagent (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega) and incubated for an additional 30 min at room temperature. LDH activity was measured using a Perkin Elmer Envision Multilabel Reader (Excitation 555 nm, Emission 590 nm) and data were normalized to 100% PMN lysis induced by 0.1% v/v Triton-X100.

Results

The ability of different mAb 5133 and the mAb-FN3 domain 5133 conjugates to neutralize the cytotoxic activity of LukED and LukAB towards human PMNs was assessed. These studies demonstrate that the FN3 domain-mAb conjugates with the LukD (PRASA A6 LC-D construct 5 and PRASA A6 HC-D construct 8) or anti-LukAB FN3 domain (PRASA A6 HC-AB construct 6) were able to neutralize the respective toxin irrespectively if the FN3 domains were appended to the light of heavy chain (PRASA A6 LC-D construct 5 vs. PRASA A6 HC-D construct 8) (FIG. 10A). Moreover, simultaneous conjugation of both the LukD and LukAB FN3 domains to the mAb 5133 (PRASA A6 LC-D HC-AB construct 7, PRASA A6 HC AB-D construct 9, and PRASA A6 HC D-AB construct 10) (FIGS. 10A-10B) also resulted in potent neutralization of the respective toxin.

Summary

These studies show that only the FN3 domain-mAb conjugates are capable of blocking LukED and LukAB killing of primary hPMNs, but not the IgG1 isotype control, A6 construct 3, or PRASA A6 construct 4 mAbs. Taken together, these data establish that the mAb-FN3 domain conjugates are functional and can block the cytotoxic activity of potent Staph toxins.

Example 12 Neutralizing Effects of the mAb-FN3 Domain Conjugates in a Model of S. aureus-Mediated Killing of Human Neutrophils Post-Phagocytosis

Among the bi-component toxins produced by Staph, LukAB is responsible for killing human neutrophils (hPMNs) during ex vivo infection with live bacteria. Staph-produced LukAB kills hPMNs by targeting the plasma membrane or the phagosomal membrane post-phagocytosis. To evaluate if the mAb-FN3 domain conjugates protect hPMNs from LukAB-mediated killing post-phagocytosis of live Staph, an ex vivo infection model was developed.

Procedure

Staph strain Newman, a highly virulent methicillin sensitive strain, was subcultured 1:100 in RPMI+CAS for 5 hours and normalized to 1×10⁷ CFU/mL in RPMI supplemented with 10 mM HEPES and 0.1% HSA (RPMI-HH). Aliquots of 1 mL of the normalized bacteria were pelleted by centrifugation and resuspended in 300 μl of different mAb 5133 or mAb-FN3 domain conjugates at 1.5 mg/ml, or PBS (non-opsonized). Samples were brought up to 1 mL with RPMI-HH and then incubated for 20 minutes at 37° C. for opsonization. The opsonized bacteria were subsequently mixed with 2×10⁵ freshly isolated primary hPMNs in RPMI-HH supplemented with EtBr at a multiplicity of infection (MOI) of ˜10:1 (bacteria:hPMN). Bacteria and cells were synchronized by centrifugation at 1500 RPM for 7 minutes and placed in a 37° C. 5% CO₂ incubator. Ethidium bromide fluorescence, as an indicator of hPMN membrane permeability, was measured during a two hour infection.

Results

The ability of different mAb 5133 and the mAb-FN3 domain 5133 conjugates to neutralize the cytotoxic activity of LukAB protecting hPMNs post Staph phagocytosis was assessed. These experiments demonstrate that the A6 construct 3 and the PRASA A6 construct 4 mAb confer minimal protection to hPMNs from S. aureus-mediated killing compared to PBS treated bacteria (no mAb). In contrast, both of the mAb-FN3 domain conjugates tested, PRASA A6 HC-AB construct 6 and PRASA A6 LC-D AB construct 7, protected hPMNs from LukAB-mediated membrane damage post-phagocytosis. No notable difference was observed between the mAb-FN3 domain conjugate containing only the anti-LukAB FN3 domains compared to that of the conjugate containing both the anti-LukAB and the anti-LukD FN3 domains (FIG. 11). This finding is consistent with the lack of LukED production during ex vivo infection models (DuMont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); Alonzo et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83:423-435 (2012); DuMont et al., “Staphylococcus aureus Elaborates Leukocidin AB to Mediate Escape From Within Human Neutrophils,” Infect. Immun. 81:1830-1841 (2013), all of which are hereby incorporated by reference in their entirety).

Summary

These experiments establish that the Staph LukAB-mediated cytotoxicity post-phagocytosis could be neutralized from within the cell by the mAb-FN3 domain conjugates.

Example 13 Neutralizing Effects of the mAb-FN3 Domain Conjugates in a Model of Neutrophil-Mediated Killing of S. aureus

LukAB kills hPMNs post-phagocytosis and promotes Staph escape and growth (DuMont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); DuMont et al., “Staphylococcus aureus Elaborates Leukocidin AB to Mediate Escape From Within Human Neutrophils,” Infect. Immun. 81:1830-1841 (2013); DuMont et al., “Staphylococcus aureus LukAB Cytotoxin Kills Human Neutrophils by Targeting the CD11b Subunit of the Integrin Mac-1,” Proc. Natl. Acad. Sci. USA 110:10794-10799 (2013), all of which are hereby incorporated by reference in their entirety). Infection of hPMNs with isogenic deletion mutant strains lacking lukAB have suggested that blocking LukAB activity diminishes bacterial growth in ex vivo OPK assays (DuMont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); DuMont et al., “Staphylococcus aureus Elaborates Leukocidin AB to Mediate Escape From Within Human Neutrophils,” Infect. Immun. 81:1830-1841 (2013); DuMont et al., “Staphylococcus aureus LukAB Cytotoxin Kills Human Neutrophils by Targeting the CD11b Subunit of the Integrin Mac-1,” Proc. Natl. Acad. Sci. USA 110:10794-10799 (2013), all of which are hereby incorporated by reference in their entirety). To investigate if the LukAB mAb-FN3 domain conjugates promote hPMN-mediated control of S. aureus growth; an OPK assay was implemented where bacterial growth was monitored by measuring GFP fluorescence.

Procedure

For these experiments the Staph strain Newman (MSSA) that contains a plasmid that expresses the green fluorescent protein (gfp) constitutively by using the sarA promoter was employed. The GFP-labeled bacteria was subcultured 1:100 in RPMI+CAS supplemented with chloramphenicol for 5 hours and normalized to 1×10⁷ CFU/mL in RPMI supplemented with 10 mM HEPES and 0.1% HSA (RPMI-HH). Aliquots of 1 mL of the normalized bacteria were pelleted by centrifugation and resuspended in 300 μl of different mAb or mAb-FN3 domain conjugates at 1.5 mg/ml, or PBS (non-opsonized). Samples were brought up to 1 mL with RPMI-HH and then incubated for 20 minutes at 37° C. for opsonization. The opsonized bacteria were subsequently mixed with 2×10⁵ freshly isolated primary hPMNs in RPMI-HH at a multiplicity of infection (MOI) of ˜10:1 (bacteria:hPMN). Bacteria and cells were synchronized by centrifugation at 1500 RPM for 7 minutes and placed in a 37° C. 5% CO₂ incubator. GFP fluorescence, as an indicator of bacterial growth was measured 5 hours post-infection.

Results

The ability of different mAb 5133 and the mAb-FN3 domain5133 conjugates to promote hPMN-mediated growth restriction of Staph was assessed. These experiments demonstrate that in comparison to the IgG1 isotype control, both A6 construct 3 and PRASA A6 construct 4 promote hPMN-mediated S. aureus growth-restriction (FIG. 12). This is consistent with the opsonic capability of the 5133 mAb. Importantly, the LukAB FN3 domain-mAb 5133 conjugates (PRASA A6 HC-AB construct 6 and PRASA A6 LC-D HC-AB construct 7) enhanced PMN-mediated growth restriction compared to the PRASA A6 parental mAb (construct 4) (FIG. 12). The lack of enhancement in growth restriction by the CR5133/anti-LukD FN3 domain-mAbs (PRASA A6 LC-D construct 5) is consistent with previous findings that LukED production is minimal during ex vivo infection models (DuMont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); Alonzo et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83:423-435 (2012); DuMont et al., “Staphylococcus aureus Elaborates Leukocidin AB to Mediate Escape From Within Human Neutrophils,” Infect. Immun. 81:1830-1841 (2013), all of which are hereby incorporated by reference in their entirety).

Summary

These experiments establish that the anti-LukAB mAb-FN3 domain conjugates neutralize LukAB, promoting hPMN-mediated Staph growth-restriction.

Example 14 In Vitro Glycosylation of Ni-NTA Resin-Bound SdrC4 Protein with S. Aureus Cell Lysates Results in an Epitope Recognized by mAb 5133

In order to further validate that SdgB-mediated glycosylation of SDR-containing proteins results in creation of a specific epitope recognized by mAb 5133, in vitro glycosylation of a representative SDR protein (recombinant SdrC4 protein purified from E. coli) was undertaken using cell lysates prepared from different S. aureus strains and the reaction products separated by SDS-PAGE and analyzed by Western blot analysis. These studies employed the USA300 parental JE2 S. aureus strain and mutants with transposon insertions that inactivate either the sdgA or sdgB glycosyltransferases (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes,” mBio 4(1):e00537-12 (2013), which is hereby incorporated by reference in its entirety) obtained from the Network on Antibiotic Resistance in Staphylococcus aureus (NARSA).

Procedure

Lysates were prepared from S. aureus strains JE2, NE381 (an sdgA transposon mutant of JE2) and NE105 (an sdgB transposon mutant of JE2). 25 mL cultures of the strains were grown in Tryptic Soy Broth (TSB) at 37° C., 200 rpm and cells harvested at an OD₆₀₀ of 0.4 to 0.6 by centrifugation at 6,000 rpm for 10 minutes. Cell pellets were re-suspended in 1 mL PBS containing 200 μg lysostaphin (Sigma L9043) and 250 units of universal nuclease (Pierce 88700). Cells were incubated at 37° C. for 45 minutes and then centrifuged at 13,000 rpm, 4° C. for 10 minutes. 750 μL of the upper supernatant were transferred to another tube and placed on ice. Recombinant SdrC4 protein purified from E. coli was bound to Ni-NTA agarose (Qiagen 1018244). Specifically, 100 μL of the Ni-NTA agarose was placed into a microfuge tube, centrifuged and washed once with 500 μL PBS containing protease inhibitors (Roche Complete EDTA-free). The resin was then re-suspended with approximately 300 μg protein and incubated on a rocking platform at 4° C. for 1.5 hours. The resin was then centrifuged at 12,000 rpm for 1 minute and washed once with 500 μL wash buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 plus protease inhibitors). The resin was then re-suspended in either 300 μl PBS or S. aureus lysate and incubated at 37° C. for 1 hour. After 1 hour, samples were centrifuged, washed three times with 500 μl wash buffer, and bound protein was eluted in 150 μL elution buffer (wash buffer containing 250 mM imidazole) for 5 minutes at room temperature.

For Western blot analysis, eluted proteins were heated at 70° C. for 10 minutes in 1×LDS sample buffer (Life Technologies NP0007) containing 100 mM DTT, and then electrophoresed on two 10% bis-tris NuPAGE gels in MOPS buffer at 150 volts for 1 hour and 40 minutes. Following electrophoresis, one gel was stained with colloidal blue (Life Technologies 46-7015 and 46-7016), and the other gel was blotted to PVDF membrane (Life Technologies IB401002) using the iBlot instrument. The PVDF blot was probed with mAb 5133 at a 1:500 dilution and subsequently detected using the FemtoMax kit (Rockland Immunochemicals KCA001).

Results

As shown in FIG. 13, incubation of resin-bound SdrC4 protein with lysates prepared from S. aureus strains JE2 (Panel B, lanes 2 and 6) and an otherwise-isogenic sdgA mutant derivative (Panel B, lanes 3 and 7) resulted in the detection of a specific ˜95 kDa species in a Western blot employing mAb 5133. In contrast no such protein band was detected by mAb 5133 following incubation of resin-bound SdrC4 protein with lysates prepared from a sdgB mutant derivative of S. aureus strain JE2 (Panel B, lanes 4 and 8) or in the control sample wherein PBS replaced cell lysate (Panel B, lanes 1 and 5). The colloidal blue stained gel shown in FIG. 13, panel A indicates that essentially equivalent amounts of the recombinant SdrC4 protein were loaded in Lanes 1-4 and 5-8.

Summary

The lack of detection of the ˜95 kDa protein band with mAb 5133 following incubation of column-bound SdrC4 protein with a lysate prepared from the sdgB mutant of strain JE2 further supports the contention that SdgB is a specific glycosyltransferase for the SdrC protein and is necessary for creation of the epitope of mAb 5133.

Example 15 In Vitro Glycosylation of SdrC4 or SdrC5 Protein with Purified SdgB Glycosyltransferase Protein Results in the Generation of an Epitope Recognized by mAb 5133

In order to further validate that SdgB-mediated glycosylation of SDR-containing proteins results in creation of a specific epitope recognized by mAb 5133, in vitro glycosylation of representative SDR proteins (SdrC4 or SdrC5) was undertaken using a recombinant form of the S. aureus SdgB glycosyltransferase and the reaction products separated by SDS-PAGE and analyzed by Western blot analysis.

Procedure

A recombinant form of the S. aureus SdgB glycosyltransferase bearing a C-terminal poly-histidine (His)₆ affinity tag (SEQ ID NO:99) was expressed in E. coli and purified via Ni-NTA affinity chromatography. For in vitro glycosylation reactions, 100 μg of recombinant SdrC4 (SEQ ID NO:100) or SdrC5 (SEQ ID NO:101) protein were incubated+/−30 μg of Uridine diphosphate N-acetylglucosamine (UDP-GlcNac)+/−4 μg of recombinant SdgB in a final volume of 100 μl 100 mM Tris pH 7.5 or 100 mM Tris pH 7.5 plus 10% glycerol at 37 C for 1 hour. For western blot analysis, reaction products were first resolved by SDS-PAGE with 1 μg of protein per lane separated on a NuPAGE 4-12% Bis-Tris Gel (NP0335BOX) at 200V constant until the dye front has reached the bottom of the gel. Proteins were then transferred to PVDF membranes (Transfer iBlot Gel Transfer Stacks PVDF IB401002) using the Invitrogen iBlot.

In FIG. 14A, panel A, the primary antibody was Construct 3 (mAb 5133 A6; SEQ ID NOs:64 HC and 65 LC) at 2 μg/ml in PBS+3% BSA and the secondary detection antibody was HRP-conjugated goat anti-human IgG (H+L) from Jackson Immunoresearch (109-036-088 lot 101421) at a 1:10,000 dilution in PBS+3% BSA Western blots. Following incubation in an HRP chemiluminescent substrate detection solution (Pierce ECL Plus), blots were exposed to X-ray film. In FIG. 14A, panel B, the primary antibody for anti-His tag detection was a HRP-conjugated anti-6× histidine mAb (MAB050H lot EGE0608041, R&D Systems) at a 1:2000 dilution in PBS+3% BSA with subsequent detection via X-ray film.

In FIG. 14B, panel A, the primary antibody was Construct 3 (mAb 5133 A6; SEQ ID NOs:64 HC and 65 LC) at 2 μg/ml in PBS+3% BSA and the secondary detection antibody was HRP-conjugated goat anti-human IgG (H+L) from Jackson Immunoresearch (109-036-088 lot 101421) at a 1:10,000 dilution in PBS+3% BSA Western blots. Following incubation in an HRP chemiluminescent substrate detection solution (Pierce ECL Plus), blots were exposed to X-ray film. The blot was then stripped using Thermo Scientific Restore Western stripping buffer and anti-his detection then undertaken using an HRP-conjugated anti-6× histidine mAb (MAB050H lot EGE0608041, R&D Systems) at a 1:2000 dilution in PBS+3% BSA with subsequent detection via X-ray film (FIG. 14B, panel B).

In FIG. 14C, panel A, the primary antibody was Construct 1 (mAb 5133; SEQ ID NOs:60 HC and 61 LC) at 2 μg/ml in Rockland fluorescent western blocking buffer (MB-070). The secondary detection employed IRDye 800CW goat anti-human IgG (H+L) Li-cor 926-32232 with detection using the Odyssey InfraRed imager. In FIG. 14C, panel B, anti-His detection employed the a HRP-conjugated anti-6× histidine mAb (MAB050H lot EGE0608041, R&D Systems) at a 1:2000 dilution in PBS +3% BSA with subsequent detection via X-ray film.

Results

As shown in FIGS. 14A, 14B, and 14C, the incubation of recombinant SdrC4 or SdrC5 proteins purified from E. coli in the presence of both SdgB and UDP-GlcNAc results in the generation of species detected by Construct 1 (mAb 5133; SEQ ID NOs:60 HC and 61 LC) and Construct 3 (mAb 5133 A6; SEQ ID NOs:64 HC and 65 LC). Detection of the (His)₆ epitopes of the SdrC4 and SdrC5 constructs in all lanes verifies that near equivalent amounts of protein were present.

Summary

These results further substantiate prior observations that mAb 5133 only detects SDR family proteins following SdgB-mediated glycosylation.

Example 16 mAb 5133 does not Specifically Bind S. aureus Lipoteichoic Acid (LTA)

The lipoteichoic acid (LTA) of S. aureus had previously been identified as the cellular target of mAb 5133 (U.S. Pat. No. 8,460,666 to Throsby et al., which is hereby incorporated by reference in its entirety). To determine if this is indeed the case, the relative binding of mAb 5133 to surface-bound S. aureus LTA and recombinant SdgB-glycosylated SdrC4 protein was determined in parallel ELISA format assays. As a positive control for LTA binding, pagibaximab (SEQ ID NO:102 HC+SEQ ID NO:103 LC), a well characterized anti-LTA mAb (Ginsburg I., “Role of Lipoteichoic Acid in Infection and Inflammation,” Lancet Infect. Dis. 2(3):171-9 (2002); Weisman et al., “Safety and Pharmacokinetics of a Chimerized Anti-Lipoteichoic Acid Monoclonal Antibody in Healthy Adults,” International Immunopharmacol. 9: 639-644 (2009), which are hereby incorporated by reference in their entirety) was included. As a negative control for binding of both SdgB-glycosylated SdrC4 protein and LTA, CNTO3930 (SEQ ID NO:104 HC+SEQ ID NO:105 LC), a mAb that specifically targets the respiratory syncytial virus F (RSV-F) glycoprotein was also included.

Procedure

High binding 96-well ELISA plates (Nunc) were coated with either SdgB-glycosylated recombinant SdrC4 protein or S. aureus lipoteichoic acid (LTA) (L 2515, Sigma) at 5 μg/mL in PBS and incubated overnight at 4° C. Plates were washed three times with ELISA wash buffer (0.15M NaCl, 0.02% Tween-20) and blocked with blocking buffer (Superblock Thermo 37515) for one hour at ambient temperature. In separate dilution plates, antibodies were serially diluted three-fold in 3% BSA-PBS blocking buffer starting at 10 μg/mL. ELISA plates were washed three times with ELISA wash buffer and antibody dilutions were transferred from the dilution plates to the ELISA plates and incubated for one hour at ambient temperature. ELISA plates were washed three times with ELISA wash buffer and a secondary goat anti-human Fc gamma-specific-HRP (Jackson Immunoresearch 109-035-098) was diluted 1:15,000 in blocking buffer and added to the plates. Plates were incubated with secondary antibody for one hour at ambient temperature then washed five times with ELISA wash buffer. TMB Substrate (Fitzgerald) was then added to the plates and allowed to develop for approximately four minutes, and then the reaction was stopped with addition of 0.5M HCl. Absorbance was read on the SpectraMax M5 Microplate Reader at 450 nm. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against each antigen.

Results

As shown in of FIG. 15, panel A, mAb 5133 exhibits potent and concentration-dependent binding to SdgB-glycosylated SdrC4 protein. In contrast, no binding to SdgB-glycosylated SdrC4 protein is detected with either pagibaximab or CNTO 3930. FIG. 15, panel B, shows the relative binding of each test article to purified S. aureus LTA, and, as expected, pagibaximab exhibited potent and concentration-dependent binding. In contrast, no binding or very weak binding to LTA is detected with CNTO 3930 or mAb 5133, respectively. The apparent weak binding of mAb 5133 to S. aureus LTA may be accounted for by contamination of the LTA preparation with native SDR proteins as it is know that preparations of LTA are often contaminated with host proteins (Silvestri et al., “Purification of lipoteichoic acids by using phosphatidyl choline vesicles,” Infection and Immunity 22(1):107-118 (1978), which is hereby incorporated by reference in its entirety).

Summary

These results suggest that mAb 5133 recognizes SdgB-glycosylated SDR family proteins and not LTA.

Example 17 mAb 5133 and Variants Bearing the PRASA and/or A6 Mutations Retain Binding to the Human and Mouse FcRn Receptors

In order to demonstrate that the PRASA and A6 mutations do not interfere significantly with the engagement of the FcRn receptor, that plays a critical role in determining the in vivo half-life of antibodies (Kuo & Aveson, “Neonatal Fc Receptor and IgG-Based Therapeutics,” mAbs 3(5): 422-30 (2011), which is hereby incorporated by reference in its entirety), mAb 5133 and variants bearing PRASA and/or A6 mutations were tested for binding to purified human and/or mouse FcRn receptors in an ELISA format, competition binding assay (Kinder et al., “Engineered Protease-Resistant Antibodies With Selectable Cell-Killing Functions,” J. Biol. Chem. 288:30843-30854 (2013), which is hereby incorporated by reference in its entirety).

Procedure

The test articles employed were mAb 5133 (SEQ ID NOs:60 HC and 61 LC), mAb 5133 PRASA (SEQ ID NOs:62 HC and 63 LC), mAb 5133 A6 (SEQ ID NOs:64 HC and 65 LC) and mAb 5133 PRASA A6 (SEQ ID NOs:66 HC and 67 LC). CNTO 3929 (SEQ ID NOs:106 HC and 107 LC), a human IgG1 antibody targeting the respiratory syncytial virus F (RSV-F) glycoprotein, was employed as the competitor mAb. A competitive binding assay was used to assess the relative affinities of different test articles to recombinant forms of human or mouse FcRn-His₆ (in which the transmembrane and cytoplasmic domains of FcRn were replaced with a poly-histidine affinity tag). Prior to use in these assays, the test articles and CNTO 3929 were dialyzed into MES buffer (0.05M MES, pH 6.0) overnight at 4° C. Ninety-six-well copper-coated plates (Thermo Scientific) were used to capture FcRn-His₆ at 5 μg/mL in phosphate buffered saline (PBS) after which plates were washed with 0.15 M NaCl, 0.02% Tween 20, pH ˜5 and then incubated with blocking reagent (0.05 M MES, 0.025% BSA, 0.001% Tween 20, pH 6.0 plus 10% ChemiBLOCKER (Millipore)). Plates were washed as previously, and then serial dilutions of competitor test antibody in blocking reagent were added to the plate in the presence of a fixed 1 μg/mL concentration of a biotinylated preparation of CNTO 3929. Plates were incubated at room temperature for 1 h, washed three times as previously, and then incubated with a 1:10,000 dilution of streptavidin-HRP (Jackson ImmunoResearch Laboratories) at room temperature for 30 min. Plates were washed five times as above, and bound streptavidin-HRP was detected by adding 3,3′,5,5′-tetramethylbenzidine peroxidase substrate with Stable Stop (Fitzgerald Industries International) and incubating for 4 min. Color development was stopped by addition of 0.5 M HCl. Optical densities were determined with a SpectraMax Plus384 plate reader (Molecular Devices) at 450-nm wavelength. Data were fitted to a sigmoidal dose-response curve using GraphPad Prism v5.

Results

As shown in FIG. 16 (panel A), variants of mAb 5133 bearing the PRASA and/or A6 mutations exhibit indistinguishable binding to the human FcRn receptor under the conditions employed in this in vitro competitive binding assay (conducted at pH 6.0). Similarly, as shown in FIG. 16 (panel B), a variant of mAb 5133 bearing both the PRASA and A6 mutations exhibits binding to the mouse FcRn receptor which is nearly identical to that of mAb 5133 under the conditions employed in this in vitro competitive binding assay.

Summary

Introduction of the PRASA and A6 mutations, alone or in combination, have minimal if any detectable impact on the affinity of variants of mAb 5133 for the human or mouse FcRn receptor in vitro.

Example 18 Fusion Proteins Bearing the PRASA and/or A6 Mutations Retain Binding to the Human FcRn Receptor

In order to demonstrate that the PRASA and A6 mutations in the context of mAb-FN3 domain fusion proteins do not interfere significantly with the engagement of the FcRn receptor, as series of fusion proteins bearing PRASA and/or A6 mutations were tested for binding to purified human FcRn receptor in an ELISA format, competition binding assay (Kinder et al., “Engineered Protease-Resistant Antibodies With Selectable Cell-Killing Functions,” J. Biol. Chem. 288:30843-30854 (2013), which is hereby incorporated by reference in its entirety).

Procedure

The test articles employed were mAb 5133 PRASA A6 (SEQ ID NOs:66 HC and 67 LC), mAb 5133 PRASA A6 HC LukAB (SEQ ID NOs:70 HC and 71 LC), mAb 5133 PRASA A6 HC LukD (SEQ ID NOs:74 HC and 75 LC), mAb 5133 PRASA A6 HC LukD HC LukAB (SEQ ID NOs:78 HC and 79 LC) and mAb 5133 PRASA A6 HC LukAB HC LukD (SEQ ID NOs:76 HC and 77 LC). CNTO 3929 (SEQ ID NOs:106 HC and 107 LC), a human IgG1 antibody targeting the respiratory syncytial virus F (RSV-F) glycoprotein, was employed as the competitor mAb. A competitive binding assay was used to assess the relative affinities of different test articles to a recombinant form of human FcRn-His₆ (in which the transmembrane and cytoplasmic domains of FcRn were replaced with a poly-histidine affinity tag). Prior to use in these assays, the test articles were dialyzed into MES buffer (0.05M MES, pH 6.0) overnight at 4° C. Ninety-six-well copper-coated plates (Thermo Scientific) were used to capture FcRn-His₆ at 5 μg/mL in phosphate buffered saline (PBS) after which plates were washed with 0.15 M NaCl, 0.02% Tween 20, pH ˜5 and then incubated with blocking reagent (0.05 M MES, 0.025% BSA, 0.001% Tween 20, pH 6.0 plus 10% ChemiBLOCKER (Millipore)). Plates were washed as previously, and then serial dilutions of competitor test antibody in blocking reagent were added to the plate in the presence of a fixed 1 μg/mL concentration of a biotinylated preparation of CNTO 3929. Plates were incubated at room temperature for 1 h, washed three times as previously, and then incubated with a 1:10,000 dilution of streptavidin-HRP (Jackson ImmunoResearch Laboratories) at room temperature for 30 min. Plates were washed five times as above, and bound streptavidin-HRP was detected by adding 3,3′,5,5′-tetramethylbenzidine peroxidase substrate with Stable Stop (Fitzgerald Industries International) and incubating for 4 min. Color development was stopped by addition of 0.5 M HCl. Optical densities were determined with a SpectraMax Plus384 plate reader (Molecular Devices) at 450-nm wavelength. Data were fitted to a sigmoidal dose-response curve using GraphPad Prism v5.

Results

As shown in FIG. 17, mAb 5133 PRASA A6-based fusion proteins bearing heavy chain appended anti-LukD and/or anti-LukAB FN3 domains exhibit binding to the human FcRn receptor under the conditions employed in this in vitro competitive binding assay (conducted at pH 6.0) with somewhat weaker binding observed for the fusion proteins compared to the parental mAb (mAb 5133 PRASA A6).

Summary

Introduction of the PRASA and A6 mutations have minimal impact on the affinity of mAb 5133-based fusion proteins for the human FcRn receptor in vitro.

Example 19 Variants of mAb 1533 Bearing the A6 Mutations are Unable to

Engage with Either of the S. aureus IgG Binding Proteins, Protein-A or Sbi.

Staphylococcus aureus expresses two known immunoglobulin binding proteins, Protein A (see Foster T. J., “Immune Evasion by Staphylococci,” Nature Reviews Microbiology 3:948-958 (2005), which is hereby incorporated by reference in its entirety, and the second binding protein for immunoglobulins (Sbi) (Zhang et al., “A Second IgG-Binding Protein in Staphylococcus aureus,” Microbiology 144:985-991 (1998), which is hereby incorporated by reference in its entirety), that are each displayed in cell-surface anchored or secreted forms (Smith et al., “The Immune Evasion Protein Sbi of Staphylococcus aureus Occurs Both Extracellularly and Anchored to the Cell Envelope by Binding to Lipotechoic Acid” Mol. Microbiol. 83:789-804 (2012); Becker et al., “Release of Protein A From the Cell Wall of Staphylococcus aureus,” Proc. Natl. Acad. Sci. (USA) 112(4):1574-9 (2014), which are hereby incorporated by reference in their entirety) and share a pair of conserved helices involved in binding to the Fc region of IgG proteins (Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008), which is hereby incorporated by reference in its entirety). In order to further demonstrate that mAb 5133 variants bearing the A6 mutations are unable to engage with either of the immunoglobulin binding proteins of Staphylococcus aureus, Western blot analysis of lysates prepared from a series of strains that express either both Protein A and Sbi or express only one of these two proteins.

Procedure

Test articles used for the Western analyses were mAb 5133 (SEQ ID NOs:60 HC and 61 LC), mAb 5133 A6 (SEQ ID NOs:64 HC and 65 LC) and mAb 5133 PRASA A6 (SEQ ID NOs:66 HC and 67 LC). Lysates were prepared from S. aureus strains JE2 (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes,” MBio. 4:e00537-12 (2013), which is hereby incorporated by reference in its entirety) and Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J. Bacteriol. 190(1):300-10 (2008), which is hereby incorporated by reference in its entirety) both wild-type for expression of both Protein A and Sbi, derivatives of JE2 NE286 (a spa transposon insertion mutant of JE2), NE453 (a sbi transposon insertion mutant of JE2) and Wood 46 (a strain known to lack expression of Protein A (Forsgen et al., “Lymphocyte Stimulation by Protein A of Staphylococcus aureus,” Eur. J. Immunol. 6:207 (1976); Ringden et al., “Activation of Human B and T Lymphocytes by Protein A of Staphylococcus aureus,” Eur. J. Immunol. 8:47 (1978), which are hereby incorporated by reference in their entirety). Briefly, cultures of the strains were grown in Tryptic Soy Broth (TSB) at 37° C., 200 rpm and cells harvested from 1 mL of culture by centrifugation at 6,000 rpm for 10 minutes following overnight growth. Cell pellets were re-suspended in 1 mL of lysostaphin buffer (50 mM Tris, pH 8.0, 20 mM MgCl2 with 1 micrograms/mL lysostaphin (Sigma L-9043)). For Western analysis, ˜30 microliters of each sample were mixed with ˜10 microliters 4× NuPAGE LDS sample buffer (Invitrogen NP0007) and electrophoresed on a 4-12% gradient NuPAGE gels. Gels were transferred to PVDF membranes and subsequently blocked in a PBS/3% milk solution for 1 hour. Western detection test articles were incubated with the blots at 2 μg/mL in Rockland fluorescent western blocking buffer with gentle agitation at room temperature (for >=2 hours) and washed four times with 1×PBS 0.5% (10 min each wash). The blots were then incubated with a secondary antibody (goat anti-human IgG (H+L) Li-co IRDye (1:10,000) for 1 hour at room temperature and then washed 4 times in with 1×PBS 0.5% (10 min each wash). Following washing, the Western blot membranes were imaged with using an Odyssey InfraRed imager.

Results

As shown in FIG. 18A, panel A, mAb 5133 detects a series of proteins (labeled C) corresponding to the glycosylated forms of Serine Aspartate Repeat (SDR) protein family members in all strains tested. For strain JE2 (Lane 1), bands migrating at approximately 55 kDa and 48 kDa are detected by mAb 5133 that correspond to Protein A and the Sbi protein, respectively. As expected, only the ˜48 kDa Sbi protein is detected by mAb 5133 in lysates prepared from two strains that lack expression of Protein A: JE2 NE286 (Lane 2) and the Wood 46 strain (Lane 4). In contrast, only the ˜55 kDa Protein A band is detected by mAb 5133 in lysates prepared from a strain that lacks expression of the Sbi protein: NE453 (Lane 3). As shown in FIG. 18B, panel B, no bands corresponding to either Protein A or the Sbi protein is detected in these same lysates by the mAb 5133 A6 test article and supports the notion that that the A6 mutations eliminate binding to both proteins. Similarly, as shown in FIG. 18B, panel C, the mAb 5133 PRASA A6 test article fails to detect to either Protein A or the Sbi protein in lysates prepared from S. aureus strains Newman or JE2 that express both of these proteins.

Summary

These results demonstrate that mAb 5133 variants bearing the A6 mutations are unable to engage with either of the immunoglobulin binding proteins of Staphylococcus aureus, Protein A or Sbi and substantiate prior observations from an ELISA format assay (Example 9, FIG. 8) wherein no binding to Protein A was detected for a series of test articles bearing the A6 mutations.

Example 20 Binding and Neutralization of γ-Hemolysins HlgAB and HlgCB and Panton-Valentine Leukocidin (PVL) by FN3 Domains

In order to demonstrate that a sub-set of FN3 domains are able to exhibit binding to, and neutralization of, multiple leukotoxins, a series of studies were done employing the γ-hemolysins HlgAB and HlgCB and Panton-Valentine leukocidin (PVL).

Procedure

Test articles included in these studies included recombinant, purified polyhistidine-tagged versions of Luk129 (SEQ ID NO:151), Luk151 (SEQ ID NO:163), Luk188 (SEQ ID NO:185), Luk357 (SEQ ID NO:339), Luk540 (SEQ ID NO:424) and Luk647 (SEQ ID NO:526). Binding of the FN3 domains to purified, recombinant leukotoxins was determined by ELISA. Briefly, 100 μl of a 5 μg/mL solution of streptavidin (in PBS) was added per well of a 96 well White Maxisorp plate (Nunc-cat#436110) and incubated overnight at 4° C. Wells were then washed 3× with TBST (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and blocked with 300 μL/well with StartingBlock T20 (Pierce cat#37543) and incubated 45-60 minutes at room temperature (RT). The plate was then washed 3 times with TBST and 0.2 μg of biotinylated versions of the leukotoxin target antigen (in 100 μL) was added to each test well and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. Test articles were diluted to 1 μM in StartingBlock T20 and 100 μL added to test wells and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. For detection of bound test articles, 1004/well of a polyclonal anti-FN3-HRP antibody diluted 1:5000 in Starting block T20 was added and the plate incubated for 45-60 min at RT with gentle shaking. The plate was then washed 3 times with TBST. To detect bound anti-FN3-HRP antibody, 10014/well of the POD Chemiluminescence substrate (Roche-cat#11582950001) was added immediately prior to reading plates and the plates read using a Paradigm or Envision reader within 15 minutes of the substrate addition.

For leukotoxin neutralization studies, the Fn3 domain test articles (4 μg per well/10 μl of 400 μg/ml) were incubated with purified, recombinant leukotoxins for 30 mins at 4° C. Freshly isolated primary human neutrophils (hPMNs, 200,000 cells in RPMI+10 mM HEPES+0.1% HSA) were added to the mixture of toxin and FN3 domain protein to a final volume of 100 μl. Ethidium bromide was then added to the cells at 1:2000 final dilution and plates were read 30 and 60 mins post toxin addition. Following 1 hour intoxication in a 37° C. CO2 incubator, 25 μl of supernatant was carefully transferred to a new plate after spinning the plate down 1500 RPM for 10 mins. Cell Titer reagent (Promega) was added to the remaining cells and incubated for 1.5 hours. The 25 μl of supernatant were mixed with equal amounts of CytoTox-ONE™ Assay reagent (Promega) that rapidly measures the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium was measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. For the neutralization experiments, the following concentrations of purified, recombinant leukotoxins were used: HlgAB 1.25 μg/mL, HlgCB 0.63 μg/mL and PVL 0.31 μg/mL.

Results

As shown in FIG. 19, the Fn3 domain variant Luk151 exhibits binding to the HlgA subunit but does not neutralize the cytolytic activity of the HlgAB leukotoxin. In contrast, Luk129 exhibits binding to the HlgA subunit and neutralizes the cytolytic activity of the HlgAB leukotoxin as exemplified by complete inhibition of LDH release.

As shown in FIG. 20, the Fn3 domain variant Luk357 exhibits binding to the HlgC subunit but does not neutralize the cytolytic activity of the HlgCB leukotoxin. In contrast, Luk188 exhibits binding to the HlgC subunit and neutralizes the cytolytic activity of the HlgCB leukotoxin as exemplified by complete inhibition of LDH release.

As shown in FIG. 21, the Fn3 domain variant Luk540 exhibits binding to the LukS-PV subunit of the PVL leukotoxin but does not neutralize the cytolytic activity of PVL. In contrast, Luk647 exhibits binding to the LukS-PV subunit of the PVL leukotoxin and neutralizes the cytolytic activity of the PVL leukotoxin as exemplified by complete inhibition of LDH release.

Summary

FN3 domains have been identified that exhibit binding to and/or neutralization of the γ-hemolysins HlgAB and HlgCB and Panton-Valentine leukocidin (PVL).

TABLE 6 Nucleic Acid and Amino Acid Sequences of the Disclosure SEQ ID NO: Type Species Description Sequence   1 PRT Artificial Tencon LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLI QYQESEKVGEAINLTVPGSERSYDLTGLKPGTE YTVSIYGVKGGHRSNPLSAEFTT   2 PRT Artificial Linker GGGGSGGGGSGGGGSGGGGS   3 PRT Artificial Tencon BC loop TAPDAA   4 PRT Artificial Tencon FG loop KGGHRSN   5 DNA Artificial POP2222 CGGCGGTTAGAACGCGGCTAC   6 DNA Artificial POP2250 CGGCGGTTAGAACGCGGCTACAATTAATAC   7 DNA Artificial DidLigRev CATGATTACGCCAAGCTCAGAA   8 DNA Artificial MDD40 AGCCGCCGCCACCGGTTAATGGTGATGGTG ATGGTGACCACCGGTGGTGAAGATCGCAGA CAG   9 DNA Artificial MDD62 AAGAAGGAGAACCGGTATGCTGCCGGCGCC GAAAAACCTGGTTGTTTCTCGTGTTACC  10 PRT S. aureus LukA HHHHHHNSAHKDSQDQNKKEHVDKSQQKD KRNVTNKDKNSTAPDDIGKNGKITKRTETVY DEKTNILQNLQFDFIDDPTYDKNVLLVKKQGS IHSNLKFESHKEEKNSNWLKYPSEYHVDFQV KRNRKTEILDQLPKNKISTAKVDSTFSYSSGG KFDSTKGIGRTSSNSYSKTISYNQQNYDTIASG KNNNWHVHWSVIANDLKYGGEVKNRNDELL FYRNTRIATVENPELSFASKYRYPALVRSGFN PEFLTYLSNEKSNEKTQFEVTYTRNQDILKNR PGIHYAPPILEKNKDGQRLIVTYEVDWKNKTV KVVDKYSDDNKPYKAG  11 PRT S. aureus LukB KINSEIKQVSEKNLDGDTKMYTRTATTSDSQK NITQSLQFNFLTEPNYDKETVFIKAKGTIGSGL RILDPNGYWNSTLRWPGSYSVSIQNVDDNNN TNVTDFAPKNQDESREVKYTYGYKTGGDFSI NRGGLTGNITKESNYSETISYQQPSYRTLLDQS TSHKGVGWKVEAHLINNMGHDHTRQLTNDS DNRTKSEIFSLTRNGNLWAKDNFTPKDKMPV TVSEGFNPEFLAVMSHDKKDKGKSQFVVHYK RSMDEFKIDWNRHGFWGYWSGENHVDKKEE KLSALYEVDWKTHNVKFVKVLNDNEKK  12 PRT S. aureus LukD MGSSHHHHHHSSGLVPAGSHMLAQHITPVSE KKVDDKITLYKTTATSDNDKLNISQILTFNFIK DKSYDKDTLVLKAAGNINSGYKKPNPKDYN YSQFYWGGKYNVSVSSESNDAVNVVDYAPK NQNEEFQVQQTLGYSYGGDINISNGLSGGLN GSKSFSETINYKQESYRTTIDRKTNHKSIGWG VEAHKIMNNGWGPYGRDSYDPTYGNELFLG GRQSSSNAGQNFLPTHQMPLLARGNFNPEFIS VLSHKQNDTKKSKIKVTYQREMDRYTNQWN RLHWVGNNYKNQNTVTFTSTYEVDWQNHTV KLIGTDSKETNPGV  13 PRT S. aureus LukE MGSSHHHHHHSSGLVPAGSHMLNTNIENIGD GAEVIKRTEDVSSKKWGVTQNVQFDFVKDK KYNKDALIVKMQGFINSRTSFSDVKGSGYELT KRMIWPFQYNIGLTTKDPNVSLINYLPKNKIE TTDVGQTLGYNIGGNFQSAPSIGGNGSFNYSK TISYTQKSYVSEVDKQNSKSVKWGVKANEFV TPDGKKSAHDRYLFVQSPNGPTGSAREYFAP DNQLPPLVQSGFNPSFITTLSHEKGSSDTSEFEI SYGRNLDITYATLFPRTGIYAERKHNAFVNRN FVVRYEVNWKTHEIKVKGHN  14 PRT Artificial Luk17 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WITYEEKFYRGEAIVLTVPGSERSYDLTGLKP GTEYKVWIVGVKGGQGSWPLSAIFTT  15 PRT Artificial Luk19 LPAPKNLVVSRVTEDSARLSWYHAIHRLNHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLAG LKPGTEYTVSIYGVLPDAFVSSNPLSAIFTT  16 PRT Artificial Luk20 LPAPKNLVVSRVTEDSARLSWYHAIHRLNHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVLPDAFVSSNPLSAIFTT  17 PRT Artificial Luk24 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIGYFELWPNGEAIVLTVPGSERSYDLTGLKP GTEYEVFIRGVKGGLYSYPLSAIFTT  18 PRT Artificial Luk8 LPAPKNLVVSRVTEDSARLSWKRKPWAPIFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVWDHAGPKYEIESNPLSAIFTT  19 PRT Artificial Luk9 LPAPKNLVVSRVTEDSARLSWDRTYSLLNYF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVGGQHPTYESNPLSAIFTT  20 PRT Artificial Luk10 LPAPKNLVVSRVTEDSARLSWAASENAFVFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVGGKLHNQFEWLSNPLSAIFT T  21 PRT Artificial Luk11 LPAPKNLVVSRVTEDSARLSWRAKPWAPKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVSAIKPGHTSNPLSAIFTT  22 PRT Artificial Luk12 LPAPKNLVVSRVTEDSARLSWRAKPWAPKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVKASEKFIESNPLSAIFTT  23 PRT Artificial Luk21 LPAPKNLVVSRVTEDSARLSWVTKPWAEYFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVWDHAGPKYEIESNPLSAIFTT  24 PRT Artificial Luk22 LPAPKNLVVSRVTEDSARLSWRAKPWAPKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYAVSIYGVKASEKFIESNPLSAIFTT  25 PRT Artificial Luk26 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIEYAEPWVWGEAIVLTVPGSERSYDLTGLKP GTEYVVFIGGVKGGHNSTPLSAIFTT  26 PRT Artificial Luk27 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFP IVYQEWQFYGEAIVLTVPGSERSYDLTGLKPG TEYLVDIYGVKGGSWSYPLSAIFTT  27 PRT Artificial Luk28 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIQYWEWWPPGEAIVLTVPGSERSYDLTGLK PGTEYGVIILGVKGGWYSNPLSAIFTT  28 PRT Artificial Luk29 LPAPKNLVVSRVTEDSARLSWDEQFVSNFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVFVPWDGFSEINYSNPLSAIFTT  29 PRT Artificial Luk30 LPAPKNLVVSRVTEDSARLSWAFNWNYFAFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVALNTGNKKSNPLSAIFTT  30 PRT Artificial Luk31 LPAPNNLVVSRVTEDSARLSWDWDKYYTNR FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVLVRDYIRAAEWYSNPLSA IFTT  31 PRT Artificial Luk32 LPAPKNLVVSRVTEDSARLSWYHENAYLLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVYDLTPEKRSSNPLSAIFTT  32 PRT Artificial Luk33 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVAFIPDEIEFSNPLSAIFTT  33 PRT Artificial Luk34 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGPKPG TEYTVSIYGVVVVPHEFEFSNPLSAIFTT  34 PRT Artificial Luk35 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVAHIPWEFEWSKPLSAIFTT  35 PRT Artificial Luk36 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVADVPDEYEFSNPLSAIFTT  36 PRT Artificial Luk37 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVVGWPLFIQSNPLSAIFTT  37 PRT Artificial Luk38 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVELIYHGWLDFVFSNPLSAIFTT  38 PRT Artificial Luk39 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVRVFYFSVEPTWFSNPLSAIFTT  39 PRT Artificial Luk40 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVSYAGEPLLWIYSNPLSAIFTT  40 PRT Artificial Luk41 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVSEVPYSEYWFSNPLSAIFTT  41 PRT Artificial Luk42 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVVWGYRLTTEHSNPLSAIFTT  42 PRT Artificial Luk43 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVNSFGRPTLNLFSNPLSAIFTT  43 PRT Artificial Luk44 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVEWLQYYGETLFSNPLSAIFTT  44 PRT Artificial Luk45 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVAWLTNAYEWEFSNPLSAIFTT  45 PRT Artificial Luk46 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIHYEESTWAGEAIVLTVPGSERSYDLTGLKP GTEYGVVIVGVKGGLKSHPLSAIFTT  46 PRT Artificial Luk47 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIQYFETTESGEAIVLTVPGSERSYDLTGLKPG TEYVVFISGVKGGPLSWPLSAIFTT  47 PRT Artificial Luk48 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIEYGEWWPTGEAIVLTVPGSERSYDLTGLKP GTEYGVLIVGVKGGFRSSPLSAIFTT  48 PRT Artificial Luk49 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIVYAEHWPAGEAIVLTVPGSERSYDLTGLKP GTEYNVTIPGVKGGKYSDPLSAIFTT  49 PRT Artificial Luk50 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIPYSEWWPVGEAIVLTVPGSERSYDLTGLKP GTEYGVYIVGVKGGTWSAPLSAIFTT  50 PRT Artificial Luk51 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIHYFESEPGGEAIVLTVPGSERSYDLTGLKPG TEYVVFIIGVKGGWSSLPLSAIFTT  51 PRT Artificial Luk52 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIQYGEAQEFGEAIVLTVPGSERSYDLTGLKP GTEYIVFITGVKGGNKSYPLSAIFTT  52 PRT Artificial Luk53 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFT IQYFEAEANGEAIVLTVPGSERSYDLTGLKPG TEYLVFIVGVKGGHSSLPLSAIFTT  53 PRT Artificial Luk54 LPAPKNLVVSRVTEDSARLSWTAPDAVFDSF RIEYSEWWPIGEAIVLTVPGSERSYDLTGLKP GTEYGVVIAGVKGGGYSVPLSAIFTT  54 PRT Artificial Luk55 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIQYFESAGEGEAIVLTVPGSERSYDLTGLKP GTEYLVFIVGVKGGVPSYPLSAIFTT  55 PRT Artificial Luk56 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIQYIELEIGEAIVLTVPGSERSYDLTGLKPGTE YGVFISGVKGGWNSYPLSAIFTT  56 PRT Artificial Luk57 LPAPKNLVVSRVTEDSARLSWTAPDAAFNSF YIEYFEWFPAGEAIVLTVPGSERSYDLTGLKP GTEYAVIIHGVKGGQRSTPLSAIFTT  57 PRT Artificial Luk58 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIEYDESAHFGEAIVLTVPGSERSYDLTGLKP GTEYIVFIYGVKGGYASIPLSAIFTT  58 PRT Artificial Luk59 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DISYNEFAWSGEAIVLTVPGSERSYDLTGLKP GTEYVVYIHGVKGGPTSYPLSAIFTT  59 PRT Artificial Luk60 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIQYWEWWPFGEAIVLTVPGSERSYDLTGLK PGTEYGVIILGVKGGFRSTPLSAIFTT  60 PRT Human CR5133 Heavy EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY Chain YMTWIRQAPGKGPEWVSHISGSGSTIYYADS VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK  61 PRT Human CR5133 Light EIVLTQSPATLSLSPGERATLSCRASQSVSGYL Chain GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  62 PRT Human CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY Heavy Chain YMTWIRQAPGKGPEWVSHISGSGSTIYYADS VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK  63 PRT Human CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL Light Chain GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  64 PRT Human CR5133 A6 EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY Heavy Chain YMTWIRQAPGKGPEWVSHISGSGSTIYYADS VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGK  65 PRT Human CR5133 A6 Light EIVLTQSPATLSLSPGERATLSCRASQSVSGYL Chain GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  66 PRT Human CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 Heavy Chain YMTWIRQAPGKGPEWVSHISGSGSTIYYADS VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGK  67 PRT Human CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 Light Chain GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  68 PRT Artificial CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 LC-D Heavy YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGK  69 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 LC-D Light GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECGGGGSGGGGS GGGGSGGGGSLPAPKNLVVSRVTEDSARLSW RAKPWAPKFDSFLIQYQESEKVGEAIVLTVPG SERSYDLTGLKPGTEYTVSIYGVKASEKFIESN PLSAIFTT  70 PRT Artificial   CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 HC-AB YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Heavy Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKGGGGSGGGGSGGGGSGGGGS MLPAPKNLVVSRVTEDSARLSWTAPDAAFDS FWITYEEKFYRGEAIVLTVPGSERSYDLTGLK PGTEYKVWIVGVKGGQGSWPLSAIFTT  71 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 HC-AB Light GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  72 PRT Artificial CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 LC-D HC-AB YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Heavy Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKGGGGSGGGGSGGGGSGGGGS MLPAPKNLVVSRVTEDSARLSWTAPDAAFDS FWITYEEKFYRGEAIVLTVPGSERSYDLTGLK PGTEYKVWIVGVKGGQGSWPLSAIFTT  73 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 LC-D HC-AB GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Light Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECGGGGSGGGGS GGGGSGGGGSLPAPKNLVVSRVTEDSARLSW RAKPWAPKFDSFLIQYQESEKVGEAIVLTVPG SERSYDLTGLKPGTEYTVSIYGVKASEKFIESN PLSAIFTT  74 PRT Artificial CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 HC-D Heavy YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSL PAPKNLVVSRVTEDSARLSWRAKPWAPKFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVKASEKFIESNPLSAIFTT  75 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 HC-D Light GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  76 PRT Artificial CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 HC AB-D YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Heavy Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKGGGGSGGGGSGGGGSGGGGS MLPAPKNLVVSRVTEDSARLSWTAPDAAFDS FWITYEEKFYRGEAIVLTVPGSERSYDLTGLK PGTEYKVWIVGVKGGQGSWPLSAIFTTGGGG SGGGGSGGGGSGGGGSLPAPKNLVVSRVTED SARLSWRAKPWAPKFDSFLIQYQESEKVGEAI VLTVPGSERSYDLTGLKPGTEYTVSIYGVKAS EKFIESNPLSAIFTT  77 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 HC AB-D GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Light Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  78 PRT Artificial CR5133 PRASA EVQLVETGGGLVKPGGSLRLSCSASRFSFRDY A6 HC D-AB YMTWIRQAPGKGPEWVSHISGSGSTIYYADS Heavy Chain VRGRFTISRDNAKSSLYLQMDSLQADDTAVY YCARGGRATSYYWVHWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPPVAGPDVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSL PAPKNLVVSRVTEDSARLSWRAKPWAPKFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVKASEKFIESNPLSAIFTTGGGG SGGGGSGGGGSGGGGSMLPAPKNLVVSRVTE DSARLSWTAPDAAFDSFWITYEEKFYRGEAIV LTVPGSERSYDLTGLKPGTEYKVWIVGVKGG QGSWPLSAIFTT  79 PRT Artificial CR5133 PRASA EIVLTQSPATLSLSPGERATLSCRASQSVSGYL A6 HC D-AB GWYQQKPGQAPRLLIYGASSRATGIPDRFSGS Light Chain GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC  80 DNA Artificial BC6 GTGACACGGCGGTTAGAACGCGGCTACAAT TAATACATAACCCCATCCCCCTGTTGACAAT TAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACA GGATCTACCATGCTGCCGGCGCCGAAAAAC CTGGTTGTTTCTGAAGTTACCGAAGACTCTC TGCGTCTGTCTTGGNNNNNNNNNNNNNNNN NNTTYGACTCTTTCCTGATCCAGTACCAGGA ATCTGAAAAAGTTGGTGAAGCGATCAACCT GACCGTTCCGGGTTCTGAACGTTCTTACGAC CTGACCGGTCTGAAACCGGGTACCGAATAC ACCGTTTCTATCTACGGTGTTCTTAGAAGCT TCCCAAAGGC  81 DNA Artificial BC7 GTGACACGGCGGTTAGAACGCGGCTACAAT TAATACATAACCCCATCCCCCTGTTGACAAT TAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACA GGATCTACCATGCTGCCGGCGCCGAAAAAC CTGGTTGTTTCTGAAGTTACCGAAGACTCTC TGCGTCTGTCTTGGNNNNNNNNNNNNNNNN NNNNNTTYGACTCTTTCCTGATCCAGTACCA GGAATCTGAAAAAGTTGGTGAAGCGATCAA CCTGACCGTTCCGGGTTCTGAACGTTCTTAC GACCTGACCGGTCTGAAACCGGGTACCGAA TACACCGTTTCTATCTACGGTGTTCTTAGAA GCTTCCCAAAGGC  82 DNA Artificial BC8 GTGACACGGCGGTTAGAACGCGGCTACAAT TAATACATAACCCCATCCCCCTGTTGACAAT TAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACA GGATCTACCATGCTGCCGGCGCCGAAAAAC CTGGTTGTTTCTGAAGTTACCGAAGACTCTC TGCGTCTGTCTTGGNNNNNNNNNNNNNNNN NNNNNNNNTTYGACTCTTTCCTGATCCAGT ACCAGGAATCTGAAAAAGTTGGTGAAGCGA TCAACCTGACCGTTCCGGGTTCTGAACGTTC TTACGACCTGACCGGTCTGAAACCGGGTAC CGAATACACCGTTTCTATCTACGGTGTTCTT AGAAGCTTCCCAAAGGC  83 DNA Artificial BC9 GTGACACGGCGGTTAGAACGCGGCTACAAT TAATACATAACCCCATCCCCCTGTTGACAAT TAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACA GGATCTACCATGCTGCCGGCGCCGAAAAAC CTGGTTGTTTCTGAAGTTACCGAAGACTCTC TGCGTCTGTCTTGGNNNNNNNNNNNNNNNN NNNNNNNNNNNTTYGACTCTTTCCTGATCC AGTACCAGGAATCTGAAAAAGTTGGTGAAG CGATCAACCTGACCGTTCCGGGTTCTGAAC GTTCTTACGACCTGACCGGTCTGAAACCGG GTACCGAATACACCGTTTCTATCTACGGTGT TCTTAGAAGCTTCCCAAAGGC  84 DNA Artificia l30mer-L17A CGGCGGTTAGAACGCGGCTACAATTAATAC ATAACCCCATCCCCCTGTTGACAATTAATCA TCGGCTCGTATAATGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGGATC TACCATGCTG  85 DNA Artificial POP2222ext CGG CGG TTA GAA CGC GGC TAC AAT TAA TAC  86 DNA Artificial LS1114 CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AAC AAC CAG GTT TTT CGG CGC CGG CAG CAT GGT AGA TCC TGT TTC  87 DNA Artificial LS1115 CCG AAG ACT CTG CCC GTC TGT CTT GG  88 DNA Artificial LS1117 CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA  89 DNA Artificial SDG10 CATGCGGTCTCTTCCGAAAAAGTTGGTGAA GCGATCGTCCTGACCGTTCCGGGT  90 DNA Artificial SDG24 GGTGGTGAAGATCGCAGACAGCGGGTTAG  91 DNA Artificial SDG28 AAGATCAGTTGCGGCCGCTAGACTAGAACC GCTGCCACCGCCGGTGGTGAAGATCGCAGA C  92 PRT Artificial TCL19 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFXI XYXEXXXXGEAIVLTVPGSERSYDLTGLKPGTE YXVXIXGVKGGXXSXPLSAIFTT; wherein “X” is an equal mixture of 18 amino acids (no cysteine or methionine)  93 PRT Artificial TCL19 C strand DSFXIXYXE, wherein “X” is an equal mixture of 18 acids (no cysteine or methionine)  94 PRT Artificial TCL19 F strand TEYXVXIXGV, wherein “X” is an equal mixture of 18 amino acids (no cysteine or methionine)  95 PRT Artificial TCL19 C strand + DSFXIXYXEXXXXGE, wherein “X” CD loop is an equal mixture of 18 amino acids (no cysteine or methionine)  96 PRT Artificial TCL19 F strand + TEYXVXIXGVKGGXXSX, wherein FG loop “X” is an equal mixture of 18 amino acids (no cysteine or methionine)  97 PRT Artificial TCL19 A strand + LPAPKXLXVXXVXXXXAXLXWXAPDAAF, AB loop + B wherein “X” is an equal strand + BC loop mixture of 18 amino acids (no cysteine or methionine)  98 PRT Artificial TCL19 E strand XYXLT, wherein “X” is an equal mixture of 18 amino acids (no cysteine or methionine)  99 PRT S. aureus SdgB MKETAAAKFERQHMDSPDLGTLVPRGSMAM NYFVGNSLGVNLTGIEKAIINRLNLFKEMGRP AQCVFLSWNRYLYRNAQNYITSSDYINMYDF FQEATYLERNEPFDWLSYWTDECHYTLKHV ENSHDFRIYDQERFLMYAHFQDPKYRILDYV NHFDSQRRKVKRDFYDVRGFLSCSRILVDKQ QTLCEFFYNPEDDTKLEKYFSYKDGKPEVQKI IVYYANKQYFFNNETELGAFFIKQLYQHGDL FFSDRNVYTAPIFNLTPESIPVVAVLHSTHIKNI DALDSSPFKNVYKAMFENLSRYRAIIVSTEQQ KLDVEKRINHTIPVVNIPVGYSETIDTPVQTLD QRSVKLISVARYSPEKQLHQQIELIKRLVSYV PKIELHMYGFGSESKKLNELIQKYGLENHVY LRGFLSNLDQEYSDAYLSLITSNMEGFSLALL ESLAHGVPVISYDIKYGPNELITSDFNGYLITK NDEDALFDKVKYVIDHPEVQQRLSKGSLAKA QQYSKASLIKQWDQFVRLILEHHHHHH 100 PRT S. aureus SdrC4 MAEHTNGELNQSKNETTAPSENKTTKKVDSR QLKDNTQTATADQPKVTMSDSATVKETSSN MQSPQNATANQSTTKTSNVTTNDKSSTTYSN ETDKSNLTQAKDVSTTPKTTTIKPRTLNRMA VNTVAAPQQGTNVNDKVHFSNIDIAIDKGHV NQTTGKTEFWATSSDVLKLKANYTIDDSVKE GDTFTFKYGQYFRPGSVRLPSQTQNLYNAQG NIIAKGIYDSTTNTTTYTFTNYVDQYTNVRGS FEQVAFAKRKNATTDKTAYKMEVTLGNDTY SEEIIVDYGNKKAQPLISSTNYINNEDLSRNMT AYVNQPKNTYTKQTFVTNLTGYKFNPNAKN FKIYEVTDQNQFVDSFTPDTSKLKDVTDQFD VIYSNDNKTATVDLMKGQTSSNKQYIIQQVA YPDNSSTDNGKIDYTLDTDKTKYSWSNSYSN VNGSSTANGDQKKYNLGDYVWEDTNKDGK QDANEKGIKGVYVILKDSNGKELDRTTTDEN GKYQFTGLSNGTYSVEFSTPAGYTPTTANVG TDDAVDSDGLTTTGVIKDADNMTLDSGFYKT PKYSLGDYVWYDSNKDGKQDSTEKGIKGVK VTLQNEKGEVIGTTETDENGKYRFDNLDSGK YKVIFEKPAGLTQTGTNTTEDDKDADGGEVD VTITDHDDFTLDNGYYEEETSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSNSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD SDSDSDSDSDSDSDSDSDNDSDSDSDSDSDA GKHTPAKPMSTVKDQHKTAKALEHHHHHH 101 PRT S. aureus SdrC5 MTPKYSLGDYVWYDSNKDGKQDSTEKGIKG VKVTLQNEKGEVIGTTETDENGKYRFDNLDS GKYKVIFEKPAGLTQTGTNTTEDDKDADGGE VDVTITDHDDFTLDNGYYEEETSDSDSDSDS DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS DSDSDSDSNSDSDSDSDSDSDSDSDSDSDSDS DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS DSDSDSDSDSDSDSDSDSDSDNDSDSDSDSDS DAGKHTPAKPMSTVKDQHKTAKALPETGLE HHHHHH 102 PRT Human Pagibaximab EVMLVESGGGLVQPKGSLKLSCAASGFTFNN Heavy Chain YAMNWVRQAPGKGLEWVARIRSKSNNYATF YADSVKDRFTISRDDSQSMLYLQMNNLKTED TAMYYCVRRGASGIDYAMDYWGQGTSLTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 103 PRT Human Pagibaximab DIVLSQSPAILSASPGEKVTMTCRASSSVNYM Light Chain HWYQQKPGSSPKPWISATSNLASGVPARFSG SGSGTSYSLTISRVEAEDAATYYCQQWSSNPP TFGGGTMLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 104 PRT Human CNTO3930 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSG Heavy Chain MGVSWIRQPPGKALEWLAHIYWDDDKRYNP SLKSRLTITKDTSKNQVVLTMTNMDPVDTAT YYCARLYGFTYGFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 105 PRT Human CNTO3930 DIVMTQSPDSLAVSLGERATINCRASQSVDYN Light Chain GISYMHWYQQKPGQPPKLLIYAASNPESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQII EDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC 106 PRT Human CNTO3929 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSG Heavy Chain MGVSWIRQPPGKALEWLAHIYWDDDKRYNP SLKSRLTITKDTSKNQVVLTMTNMDPVDTAT YYCARLYGFTYGFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 107 PRT Human CNTO3929 DIVMTQSPDSLAVSLGERATINCRASQSVDYN Light Chain GISYMHWYQQKPGQPPKLLIYAASNPESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQII EDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSLPVTKSFNRGEC 108 PRT S. aureus HlgA NSAHHHHHHGSENKIEDIGQGAEIIKRTQDITS KRLAITQNIQFDFVKDKKYNKDALVVKMQG FISSRTTYSDLKKYPYIKRMIWPFQYNISLKTK DSNVDLINYLPKNKIDSADVSQKLGYNIGGNF QSAPSIGGSGSFNYSKTISYNQKNYVTEVESQ NSKGVKWGVKANSFVTPNGQVSAYDQYLFA QDPTGPAARDYFVPDNQLPPLIQSGFNPSFITT LSHERGKGDKSEFEITYGRNMDATYAYVTRH RLAVDRKHDAFKNRNVTVKYEVNWKTHEV KIKSITPK 109 PRT S. aureus HlgB NSAHHHHHHGSEGKITPVSVKKVDDKVTLY KTTATADSDKFKISQILTFNFIKDKSYDKDTL VLKATGNINSGFVKPNPNDYDFSKLYWGAK YNVSISSQSNDSVNVVDYAPKNQNEEFQVQN TLGYTFGGDISISNGLSGGLNGNTAFSETINYK QESYRTTLSRNTNYKNVGWGVEAHKIMNNG WGPYGRDSFHPTYGNELFLAGRQSSAYAGQ NFIAQHQMPLLSRSNFNPEFLSVLSHRQDGAK KSKITVTYQREMDLYQIRWNGFYWAGANYK NFKTRTFKSTYEIDWENHKVKLLDTKETENN K 110 PRT S. aureus HlgC NSAHHHHHHGSANDTEDIGKGSDIEIIKRTED KTSNKWGVTQNIQFDFVKDKKYNKDALILK MQGFISSRTTYYNYKKTNHVKAMRWPFQYN IGLKTNDKYVSLINYLPKNKIESTNVSQTLGY NIGGNFQSAPSLGGNGSFNYSKSISYTQQNYV SEVEQQNSKSVLWGVKANSFATESGQKSAFD SDLFVGYKPHSKDPRDYFVPDSELPPLVQSGF NPSFIATVSHEKGSSDTSEFEITYGRNMDVTH AIKRSTHYGNSYLDGHRVHNAFVNRNYTVK YEVNWKTHEIKVKGQN 111 PRT S. aureus LukF-PV NSAHHHHHHGSAQHITPVSEKKVDDKITLYK TTATSDSDKLKISQILTFNFIKDKSYDKDTLIL KAAGNIYSGYTKPNPKDTISSQFYWGSKYNIS INSDSNDSVNVVDYAPKNQNEEFQVQQTVG YSYGGDINISNGLSGGGNGSKSFSETINYKQE SYRTSLDKRTNFKKIGWDVEAHKIMNNGWG PYGRDSYHSTYGNEMFLGSRQSNLNAGQNFL EYHKMPVLSRGNFNPEFIGVLSRKQNAAKKS KITVTYQREMDRYTNFWNQLHWIGNNYKDE NRATHTSIYEVDWENHTVKLIDTQSKEKNPM S 112 PRT S. aureus LukS-PV NSAHHHHHHGSDNNIENIGDGAEVVKRTEDT SSDKWGVTQNIQFDFVKDKKYNKDALILKM QGFINSKTTYYNYKNTDHIKAMRWPFQYNIG LKTNDPNVDLINYLPKNKIDSVNVSQTLGYNI GGNFNSGPSTGGNGSFNYSKTISYNQQNYISE VERQNSKSVQWGIKANSFITSLGKMSGHDPN LFVGYKPYSQNPRDYFVPDNELPPLVHSGFN PSFIATVSHEKGSGDTSEFEITYGRNMDVTHA TRRTTHYGNSYLEGSRIHNAFVNRNYTVKYE VNWKTHEIKVKGHN 113 PRT Artificial Luk82 LPAPKNLVVSRVTEDSARLSWSNRAITTFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVEYRFRPKYTGSNPLSAIFTT 114 PRT Artificial Luk83 LPAPKNLVVSRVTEDSARLSWFRPSEDISSFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVAFPTDAKSNPLSAIFTT 115 PRT Artificial Luk85 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HINYNEEALVGEAIVLTVPGSERSCDLTGLKP GTEYGVEIEGVKGGPWSWPLSAIFTT 116 PRT Artificial Luk86 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIRYNEIDLHGEAIVLTVPGSERSYDLTGLKP GTEYQVPIAGVKVCIISKPLSAIFTT 117 PRT Artificial Luk87 LPAPKNLVVSRVTEDSARLSWANTEPSYFAF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVVWVTWGKSNPLSAIFTT 118 PRT Artificial Luk88 LPAPKNLVVSRVTEDSARLSWTLEWSLIFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVQRSVAWYFLLLASNPLSAIFT T 119 PRT Artificial Luk90 LPAPKNLVVSRVTEDSARLSWRTYPTLFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVWPRNIQPWSNPLSAIFTT 120 PRT Artificial Luk92 LPAPKNLVVSRVTEDSARLSWKRVKWVSYQ FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVASIDETVGVSNPLSAIFT T 121 PRT Artificial Luk93 LPAPKNLVVSRVTEDSARLSWWRRISRFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVDREVYDEWSSNPLSAIFTT 122 PRT Artificial Luk94 LPAPKNLVVSRVTEDSARLSWYRRFLLFIFFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVGEQWGEASDLSNPLSAIFT T 123 PRT Artificial Luk95 LPAPKNLVVSRVTEDSARLSWQHSQYFVLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVLYRQWRDSNPLSAIFTT 124 PRT Artificial Luk96 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVEHWPSWWHLNFSNPLSAIFTT 125 PRT Artificial Luk97 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVIDIIHINSWNDHSNPLSAIFTT 126 PRT Artificial Luk98 LPAPKNLVVSRVTEDSARLSWNRHSHEFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVRIWVLKLNESNPLSAIFTT 127 PRT Artificial Luk99 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIIYYERHDNGEAIVLTVPGSERSYDLTGLKP GTEYLVWIPGVKGGLTSWPLSAIFTT 128 PRT Artificial Luk100 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PITYGEYRSVGEAIVLTVPGSERSYDLTGLKP GTEYIVDIYGVKGGLFSYPLSAIFTT 129 PRT Artificial Luk101 LPAPKNLVVSRVTEDSARLSWDTEPEWFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVQRVEIRALYRSYSNPLSAIFTT 130 PRT Artificial Luk102 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVSHRFWKHVYFYSNPLSAIFTT 131 PRT Artificial Luk103 LPAPKNLVVSRVTEDSARLSWIIGLSRFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGT EYTVSIYGVDFAHQDFFRGYASNPLSAIFTT 132 PRT Artificial Luk104 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVQWWVVAFHHAPSNPLSAIFT T 133 PRT Artificial Luk106 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVAWIFTKVLNASNPLSAIFTT 134 PRT Artificial Luk107 LPAPKNLVVSRVTEDSARLSWKGPNSPPSQF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWKWRTRAHSNPLSAIFTT 135 PRT Artificial Luk108 LPAPKNLVVSRVTEDSARLSWFYYYLGKFGF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVVNWRWWPDDSNPLSAIF TT 136 PRT Artificial Luk109 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVIINRFWAWYLASSNPLSAIFTT 137 PRT Artificial Luk110 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVGFPTFLNYWQFGSNPLSAIFT T 138 PRT Artificial Luk112 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYYEFRHHGEAIVLTVPGSERSYDLTGLKP GTEYAFWIYGVKGGGSSWPLSAIFTT 139 PRT Artificial Luk113 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIEYHEEYETGEAIVLTVPGSERSYDLTGLKP GTEYWVWIAGVKGGKWSWPLSAIFTT 140 PRT Artificial Luk114 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYQELPQKGEAIVLTVPGSERSYDLTGLKP GTEYVVWIWGVKGGLTSDPLSAIFTT 141 PRT Artificial Luk116 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYQEYPAFGEAIVLTVPGSERSYDLTGLKP GTEYIVWIWGVKGGWTSWPLSAIFTT 142 PRT Artificial Luk117 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYQETISVGEAIVLTVPGSERSYDLTGLKPG TEYWVLIWGVKGGAASDPLSAIFTT 143 PRT Artificial Luk119 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIDYFEEYQKGEAIVLTVPGSERSYDLTGLKP GTEYWVWIFGVKGGIRSWPLSAIFTT 144 PRT Artificial Luk120 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EINYWEAYIHGEAIVLTVPGSERSYDLTGLKP GTEYWVWIHGVKGGGNSYPLSAIFTT 145 PRT Artificial Luk122 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYYEFAIKGEAIVLTVPGSERSYDLTGLKPG TEYAVWIYGVKGGNSSWPLSAIFTT 146 PRT Artificial Luk123 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIDYFEEYSHGEAIVLTVPGSERSYDLTGLKP GTEYWVWINGVKGGIYSYPLSAIFTT 147 PRT Artificial Luk124 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIDYVESYALGEAIVLTVPGSERSYDLTGLKP GTEYWVWIWGVKGGSLSYPMSAIFTT 148 PRT Artificial Luk125 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIIYYEHHNFGEAIVLTVPGSERSYDLTGLKPG TEYAVPIPGVKGGWQSLPLSAIFTT 149 PRT Artificial Luk126 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIDYQEWPSVGEAIVLTVPGSERSYDLTGLKP GTEYSVFIHGVKGGWLSKPLSAIFTT 150 PRT Artificial Luk128 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIQYFEEYAIGEAIVLTVPGSERSYDLTGLKPG TEYWVWISGVKGGNFSKPLSAIFTT 151 PRT Artificial Luk129 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NINYPEEFHGGEAIVLTVPGSERSYDLTGLKP GTEYEVWIWGVKGGSSSNPLSAIFTT 152 PRT Artificial Luk130 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIHYLEWEVNGEAIVLTVPGSERSYDLTGLKP GTEYIVEIWGVKGGYSSWPLSAIFTT 153 PRT Artificial Luk132 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIEYWEWDPVGEAIVLTVPGSERSYDLTGLK PGTEYPVFISGVKGGYPSVPLSAIFTT 154 PRT Artificial Luk133 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IHYGEGPEFGEAIVLTVPGSERSYDLTGLKPG TEYSVHIPGVKGGWLSWPLSAIFTT 155 PRT Artificial Luk134 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIVYLEWVVLGEAIVLTVPGSERSYDLTGLKP GTEYIVDIYGVKGGWTSRPLSAIFTT 156 PRT Artificial Luk136 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIDYFEEYVVGEAIVLTVPGSERSYDLTGLKP GTEYWVCIVGVKGGTPSPPLSAIFTT 157 PRT Artificial Luk138 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIWYQEFEVRGEAIVLTVPGSERSYDLTGLKP GTEYDVEIWGVKGGSHSWTLSAIFTT 158 PRT Artificial Luk139 LPAPKNLVVSRVTEDSARLSWTAPDAAFNSF EIHYGEWEYGGEAIVLTVPGSERSYDLTGLKP GTEYTVWIYGVKGGDSSWPLSAIFTT 159 PRT Artificial Luk140 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYQETKKSGEAIVLTVPGSERSYDLTGLKP GTEYWVLIWGVKGGTASNPLSAIFTT 160 PRT Artificial Luk143 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIQYDERTEEGEAIVLTVPGSERSYDLTGLKP GTEYFVTIPGVKGGWYSWPLSAIFTT 161 PRT Artificial Luk144 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIDYFEEWVNGEAIVLTVPGSERSYDLTGLK PGTEYWVWIQGVKGGVHSPPLSAIFTT 162 PRT Artificial Luk148 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYQELNRVGEAIVLTVPGSERSYDLTGLKP GTEYWVLIWGVKGGDSSEPLSAIFTT 163 PRT Artificial Luk151 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GITYTEVYWWGEAIVLTVPGSERSYDLTGLK PGTEYTVTIPGVKGGWISAPLSAIFTT 164 PRT Artificial Luk155 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIRYFEFIKPGEAIWLGVPGSERSYDLTGLKPG TEYHVQIRGVKGGRESYPLWADFTT 165 PRT Artificial Luk156 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIQYHETRYSGEAIWLWVPGSERSYDLTGLK PGTEYSVYIPGVKGGNVSFPLKAHFTT 166 PRT Artificial Luk158 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AISYKESGRIGEAISLIVPGSERSYDLTGLKPG TEYWVYINGVKGGITSFPLNAWFTT 167 PRT Artificial Luk159 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIDYKETGYTGEAIELEVPGSEHSYDLTGLKP GTEYFVTIGGVKGGYSSWPLVALFTT 168 PRT Artificial Luk160 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIWYTENPSLGEAIKLSVPGSERSYDLTGLKP GTEYVVEIWGVKGGRGSVPLFAIFTT 169 PRT Artificial Luk163 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIEYWEPTSDGEAIALNVPGSERSYDLTGLKP GTEYFVEIWGVKGGPRSPPLSAWFTT 170 PRT Artificial Luk164 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIEYGEPEKIGEAIWLTVPGSERSYDLTGLKP GTEYWVFIYGVKGGALSRPLTATSTT 171 PRT Artificial Luk166 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIQYFEIQPWGEAILLDVPGSERSYDLTGLKP GTEYSVIIWGVKGGPKSQPLYAWFTT 172 PRT Artificial Luk167 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIIYSEDTIPGEAIVLWVPGSERSYDLTGLKPG TEYYVQIEGVKGGHESFPLVANFTT 173 PRT Artificial Luk174 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIEYGEPEKIGEAIWLTVPGSERSYDLTGLKP GTEYWVFIYGVKGGALSRPLTATFTT 174 PRT Artificial Luk176 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIYYHEFPYGGEAIVLTVPGSERSYDLTGLKP GTEYYVRILGVKGGGLSYPLSAIFTT 175 PRT Artificial Luk177 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIYYREWGSGEAIVLTVPGSERSYDLTGLKPG TEYLVITGVKGGNPSYPLSAIFTT 176 PRT Artificial Luk178 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIHYFEAYAGGEAIVLTVPGSERSYDLTGLKP GTEYWVWIFGVKGGLYSYPLSAIFTT 177 PRT Artificial Luk179 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EINYFEAWDGGEAIVLTVPGSERSYDLTGLKP GTEYWVWISGVKGGRYSYPLSAIFTT 178 PRT Artificial Luk180 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EIHYYEPIYVGEAIVLTVPGSERSYDLTGLKPG TEYIVWIYGVKGGYSSWPLSAIFTT 179 PRT Artificial Luk182 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YILYIENDWQGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGWISPPLSAIFTT 180 PRT Artificial Luk183 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIWYWEFLHNGEAIVLTVPGSERSYDLTGLKP GTEYFVEIYGVKGGSVSVPLSAIFTT 181 PRT Artificial Luk184 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYHELNTAGEAIVLTVPGSERSYDLTGLKPG TEYLVIIHGVKGGPISSPLSAIFTT 182 PRT Artificial Luk185 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIVYREWFHYGEAIVLTVPGSERSYDLTGLKP GTEYYVVIHGVKGGYISKPLSAIFTT 183 PRT Artificial Luk186 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HITYTEYSFVGEAIVLTVPGSERSYDLTGLKP GTEYFVEIYGVKGGFISSPLSAIFTT 184 PRT Artificial Luk187 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RILYFEYKRLGEAIVLTVPGSERSYDLTGLKP GTEYFVGIHGVKGGYISRPLSAIFTT 185 PRT Artificial Luk188 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIHYWEFNPAGEAIVLTVPGSERSYDLTGLKP GTEYFVGIHGVKGGGISWPLSAIFTT 186 PRT Artificial Luk189 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSL KIFYFEFIYLGEAIVLTVPGSERSYDLTGLKPG TEYHVTIHGVKGGTISLPLSAIFTT 187 PRT Artificial Luk190 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYYEFSNYGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGQISVPLSAIFTT 188 PRT Artificial Luk191 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYWEWYHGEAIVLTVPGSERSYDLTGLKP GTEYNVVIHGVKGGYISLPLSAIFTT 189 PRT Artificial Luk192 LPAPKNLDVSRVTEDSARLSWTAPDAAFDSF VIFYYEEKPIGEAIVLTVPGSERSYDLTGLKPG TEYFVEIYGVKGGYISNPLSAIFTT 190 PRT Artificial Luk193 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYHETRPLGEAIVLTVPGSERSYDLTGLKP GTEYLVAIYGVKGGYISLPLSAIFTT 191 PRT Artificial Luk194 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYWEFSDNGEAIVLTVPGSERSYDLTGLKP GTEYLVGIYGVKGGQISQPLSAIFTT 192 PRT Artificial Luk195 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIIYYEYPAGGEAIVLTVPGSERSYDLTGLKP GTEYHVIIHGVKGGFVSVPLSAIFTT 193 PRT Artificial Luk196 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIIYIENPYWGEAIVLTVPGSERSYDLTGLKPG TEYFVIIHGVKGGYISEPLSAIFTT 194 PRT Artificial Luk197 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYWEVQANGEAIVLTVPGSERSYDLTGLKP GTEYVVGIYGVKGGYISLPLSAIFTT 195 PRT Artificial Luk198 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYTEEKTWGEAIVLTVPGSERSYDLTGLKP GTEYFVWIHGVKGGWISAPLSAIFTT 196 PRT Artificial Luk199 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYAEHSNKGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGFISWPLSAIFTT 197 PRT Artificial Luk201 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYLEHNDEGEAIVLTVPGSERSYDLTGLKP GTEYWVAIHGVKGGYISQPLSAIFTT 198 PRT Artificial Luk202 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIWYHETWRGEAIVLTVPGSERSYDLTGLKP GTEYPVVIHGVKGGFISTPLSAIFTT 199 PRT Artificial Luk203 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYVEYETWGEAIVLTVPGSERSYDLTGLKP GTEYIVAIHGVKGGYISIPLSAIFTT 200 PRT Artificial Luk204 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIIYWELWSIGEAIVLTVPGSERSYDLTGLKPG TEYFVEIYGVKGGTISTPLSAIFTT 201 PRT Artificial Luk205 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SILYWEWVANGEAIVLTVPGSERSYDLTGLK PGTEYFVEIYGVKGGWLSLPLSAIFTT 202 PRT Artificial Luk206 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYFEQFSRGEAIVLTVPGSERSYDLTGLKPG TEYFVAIHGVKGGFVSRPLSAIFTT 203 PRT Artificial Luk208 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIWYWEWYHLGEAIVLTVPGSELSYDLTGLK PGTEYWVEIYGVKGGFISQPLSAIFTT 204 PRT Artificial Luk210 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYFEYLGNGEAIVLTVPGSERSYDLTGLKP GTEYFVGIHGVKGGVISTPLSAIFTT 205 PRT Artificial Luk211 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYFEWKRLGEAIVPTVPGSERSYDLTGLKPG TEYWVGIYGVKGGPISVPLSAIFTT 206 PRT Artificial Luk212 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYTEREFYGEAIVLTVPGSERSYDLTGLKPG TEYWVGIYGVKGGNISEPLSAIFTT 207 PRT Artificial Luk213 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYHETDAYGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGFISSPLSAIFTT 208 PRT Artificial Luk214 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIHYWEYDANGEAIVLTVPGSERSYDLTGLK PGTEYLVAIYGVKGGLISVPLSAIFTT 209 PRT Artificial Luk215 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYHESVTNGEAIVLTVPGSERSYDLTGLKP GTEYLVGIYGVKGGYISDPLSAIFTT 210 PRT Artificial Luk216 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIHYFEESITGEAIVLTVPGSERSYDLTGLKPG TEYFVAIYGVKGGSISDPLSAIFTT 211 PRT Artificial Luk218 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIIYWEYRWQGEAIVLTVPGSERSYDLTGLKP GTEYIVPIHGVKGGEISPPLSAIFTT 212 PRT Artificial Luk219 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIWYWVYRTSGEAIVLTVPGSERSYDLTGLK PGTEYFVAIHGVKGGEISVPLSAIFTT 213 PRT Artificial Luk220 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYWEESPPGEAIVLTVPGSERSYDLTGLKP GTEYLVAIYGVKGGYISLPLSAIFTT 214 PRT Artificial Luk221 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYHELEHHGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGQISWPLSAIFTT 215 PRT Artificial Luk222 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SILYWEEEFGEAIVLTVPGSERSYDLTGLKPG TEYFVAIHGVKGGYISRPLSAIFTT 216 PRT Artificial Luk223 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VILYWEEENQGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGHISEPLSAIFTT 217 PRT Artificial Luk224 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYTEHGVSGEAIVLTVPGSERSYDLTGLKP GTEYWVPIHGVKGGTISQPLSAIFTT 218 PRT Artificial Luk225 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYHEFLTIGEAIVLTVPGSERSYDLTGLKPG TEYIVAIYGVKGGQISDPLSAIFTT 219 PRT Artificial Luk226 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYAEWHLDGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGYISEPLSAIFTT 220 PRT Artificial Luk227 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIYYHEWQATGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGWISSPLSAIFTT 221 PRT Artificial Luk228 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIPYYEYAVFGEATVLTVPGSERSYDLTGLKP GTEYHVIIHGVKGGYISLPLSAIFTT 222 PRT Artificial Luk229 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIFYLEWNQIGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGFISDPLSAIFTT 223 PRT Artificial Luk230 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IFYTESHFYGEAIVLTVPGSERSYDLTGLKPGT EYWVAIYGVKGGEFSFPLSAIFTT 224 PRT Artificial Luk231 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RILYWEYVTAGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGYISIPLSAIFTT 225 PRT Artificial Luk233 LPAPKNLVVSRVTEDSACLSWTAPDAAFDSF AIQYWEYSGIGEAIVLTVPGSERSYGLTGLKP GTEYFVGIAGVKGGWISLPLSAIFTT 226 PRT Artificial Luk235 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIIYHEWDKNGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGYISRPLSAIFTT 227 PRT Artificial Luk236 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYWEYILPGEAIVLTVPGSERSYDLTGLKP GTEYWVIIHGVKGGHISDPLSAIFTT 228 PRT Artificial Luk237 MLPPPKNLVVSRVTEDSARLSWTAPDAAFDS FQIIYWEYAETGEAIVLTVPGSERSYDLTGLK PGTEYIVIIHGVKGGEISRPLSAIFTT 229 PRT Artificial Luk238 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYHETVKSGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKRGQISEPLSAIFTT 230 PRT Artificial Luk239 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HISYWEYAVYGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGWISSPLSAIFTT 231 PRT Artificial Luk240 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYDEEAHNGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGTISIPLSAIFTT 232 PRT Artificial Luk241 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIYYFESYAVGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGWISWPLSAIFTT 233 PRT Artificial Luk242 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYHETEVDGEAIVLTVPGSERSYDLTGLKP GTEYVVIIHGVKGGFISYPLSAIFTT 234 PRT Artificial Luk243 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIMYFEFQALGEAIVLTVPGSERSYDLTGLKP GTEYLVLIHGVKGGLISPPLSAIFTT 235 PRT Artificial Luk244 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIYYWEFLENGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGFISWPLSAIFTT 236 PRT Artificial Luk245 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYWEFRPGGEAIVLTVPGSERSYDLTGLKP GTEYFVAIFGVKGGSISVPLSAIFTT 237 PRT Artificial Luk246 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIHYFEASPPGEAIVLTVPGSERSYDLTGLKPG TEYYVVIYGVKGGYISPPLSAIFTT 238 PRT Artificial Luk247 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYHEYVQVGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGQISDPLSAIFTT 239 PRT Artificial Luk248 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIIYWEYVDVGEAIVLTVPGSERSYDLTGLKP GTEYLVPIYGVKGGLISEPLSAIFTT 240 PRT Artificial Luk249 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIVYWEQKFYGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGFISLPLSAIFTT 241 PRT Artificial Luk250 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYDEWRGVGEAIVLTVPGSERSYDLTGLKP GTEYFVPIQGVKGGYVSDPLSAIFTT 242 PRT Artificial Luk251 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIHYHEYQQIGEAIVLTVPGSERSYDLIGLKPG TEYFVAIYGVKGGFISQPLSAIFTT 243 PRT Artificial Luk252 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYLEWPAKGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGWISPPLSAIFTT 244 PRT Artificial Luk253 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIVYWEYNPVGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGNISKPLSAIFTT 245 PRT Artificial Luk254 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIFYLEHDWRGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGSISVPLSAIFTT 246 PRT Artificial Luk255 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYWEYEQQGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGEISQPLSAIFTT 247 PRT Artificial Luk257 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYIEHVDWGEAIVLTVPGSERSYDLTGLKPG TEYFVEIYGVKGGKISAPLSAIFTT 248 PRT Artificial Luk258 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIYYFESVDWGEAIVLTVPGSERSYDLTGLKP GTEYYVYIYGVKGGWISVPLSAIFTT 249 PRT Artificial Luk259 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIIYWESQYWGEAIVLTVPGSERSYDLTGLKP GTEYIVVIHGVKGGGISDPLSAIFTT 250 PRT Artificial Luk260 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIIYYEWESAGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGFISFPLSAIFTT 251 PRT Artificial Luk261 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYWEFQKKGEAIVLTVPGSERSYDLTGLKP GTEYIVIIYGVKGGFISPPLSAIFTT 252 PRT Artificial Luk262 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYLEKTNYGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGPISGPLSAIFTT 253 PRT Artificial Luk263 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIWYWEYVRNGEAIVLTVPGSERSYDLTGLK PGTEYFVPIYGVKGGDTSPPLSAIFTT 254 PRT Artificial Luk264 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYHEYFTVGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGLISAPLSAIFTT 255 PRT Artificial Luk265 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIIYFENEYGGEAIVLTVPGSERSYDLTGLKPG TEYFVAIYGVKGGYLSVPLSAIFTT 256 PRT Artificial Luk266 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIGYLENPWDGEAIVLTVPGSERSYDLTGLKP GTEYFVFIYGVKGGHISNPLSAIFTT 257 PRT Artificial Luk267 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IHYFEYEPPGEAIVLTVPGSERSYDLTGLKPGT EYFVGIYGVKGGWVSEPLSAIFTT 258 PRT Artificial Luk268 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYPEYSARGEAIVLTVPGSERSYDLTGLKPG TEYFVVIHGVKGGFVSEPLSAIFTT 259 PRT Artificial Luk269 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIIYWEYEVAGEAIVLTVPGSERSYDLTGLKP GTEYFVSIQGVKGGAISPPLSAIFTT 260 PRT Artificial Luk270 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIVYFEHPSYGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGEISAPLSAIFTT 261 PRT Artificial Luk271 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIVYFEWAANGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGAISLPLSAIFTT 262 PRT Artificial Luk272 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYWEDTLKGEAIVLTVPGSERSYDLTGLKP GTEYVVAIHGVKGGTISHPLSAIFTT 263 PRT Artificial Luk273 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VINYWEFQPAGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGQISKPLSAIFTT 264 PRT Artificial Luk274 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYWELVWNGEAIVLTVPGSERSYDLTGLKP GTEYCVPIHGVKGGLISPPLSAIFTT 265 PRT Artificial Luk275 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYEEWQVGGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGAISQPLSAIFTT 266 PRT Artificial Luk276 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIHYFEYEIRGEAIVLTVPGSERSYDLTGLKPG TEYFVSIYGVKGGLISSPLSAIFTT 267 PRT Artificial Luk277 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYLEYDQGGEAIVLTVPGSERSYDLTGLKP GTEYLVTIHGVKGGYISEPLSAIFTT 268 PRT Artificial Luk278 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYWEFAVSGEAIVLTVPGSERSYDLTGLKP GTEYSVVIHGVKGGVISEPLSAIFTT 269 PRT Artificial Luk279 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIIYFEFFIGGEAIVLTVPGSERSYDLTGLKPGT EYFVVIHGVKGGDLSAPLSAIFTT 270 PRT Artificial Luk280 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIIYWEFASNGEAIVLTVPGSERSYDLTGLKPG TEYFVAIYGVKGGEISNPLSAIFTT 271 PRT Artificial Luk281 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYFEFQTHGEAIVLTVPGSERSYDLTGLKPG TEYFVPITGVKGGWYSDPLSAIFTT 272 PRT Artificial Luk282 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IIYWEYRTCGEAIVLTVPGSERSYDLTGLKPG TEYFVEIYGVKGGNTSPPLSAIFTT 273 PRT Artificial Luk283 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSIT IHYFEPHTGGEAIVLTVPGSERSYDLTGLKPG TEYFVPIYGVKGGYISQPLSAIFTT 274 PRT Artificial Luk285 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PILYWENITTGEAIVLTVPGSERSYDLTGLKPG TEYFVVIHGVKGGFISGPLSAIFTT 275 PRT Artificial Luk286 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYWEFQAAGEAIVLTVPGSERSYDLTGLKP GTEYFVEIYGVKGGWTSFPLSAIFTT 276 PRT Artificial Luk287 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIVYWEWQCNGEAIVLTVPGSERSYDLTGLK PGTEYFVFIHGVKGGITSAPLSAIFTT 277 PRT Artificial Luk288 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIHYWEPQGIGEAIVLTVPGSERSYDLTGLKP GTEYFVGIHGVKGGWISFPLSAIFTT 278 PRT Artificial Luk289 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IPYWEYQYAGEAIVLTVPGSERSYDLTGLKPG TEYWVGIYGVKGGSISEPLSAIFTT 279 PRT Artificial Luk290 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYIEPQFEGEAIVLTVPGSERSYDLTGLKPG TEYIVVIHGVKGGYISKPLSAIFTT 280 PRT Artificial Luk291 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIIYWEYDPHGEAIVLTVPGSERSYDLTGLKP GTEYIVSIYGVKGGYISPPLSAIFTT 281 PRT Artificial Luk292 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIEYWEWIDKGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGYISWPLSAIFTT 282 PRT Artificial Luk293 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SILYHEWSGWGEAIVLTVPGSERSYDLTGLKP GTEYFVFIHGVKGGYISPPLSAIFTT 283 PRT Artificial Luk294 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QILYWETAKSGEAIVLTVPGSERSYDLTGLKP GTEYFVEIYGVKGGWISWPLSAIFTT 284 PRT Artificial Luk296 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYYEFKYQGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGFISPPLSAIFTT 285 PRT Artificial Luk298 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYLEGNWSGEAIVLTVPGSERSYDLTGLKP GTEYFVSIYGVKGGFISEPLSAIFTT 286 PRT Artificial Luk299 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF KIFYWEWPHSGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGWISKPLSAIFTT 287 PRT Artificial Luk300 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TISYWEYAGYGEAIVLTVPGSERSYDLTGLKP GTEYFVGIHGVKGGWISKPLSAIFTT 288 PRT Artificial Luk301 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HISYWEYYARGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGVISPPLSAIFTT 289 PRT Artificial Luk302 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF KIWYLETGFRGEAIVLTVPGSERSYDLTGLKP GTEYFVPIYGVKGGYISQPLSAIFTT 290 PRT Artificial Luk303 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIQYWEWNLGGEAIVLTVPGSERSYDLTGLK PGTEYFVAIYGVKGGAISDPLSAIFTT 291 PRT Artificial Luk304 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYTELYEFGEAISLLVPGSERSYDLTGLKP GTEYSVAIAGVKGGAYSHPLHALFTT 292 PRT Artificial Luk305 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIKYAEHVVWGEAIDLLVPGSERSYDLTGLK PGTEYEVGIAGVKGGTVSVPLSARFTT 293 PRT Artificial Luk306 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI ILYDEIWPSGEAISLGVPGSERSYDLTGLKPGT EYFVAIHGVKGGNISDPLDAKFTT 294 PRT Artificial Luk307 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYTESRYYGEAIDLLVPGSERSYDLTGLKP GTEYHVRISGVKGGAFSTPLWAAATT 295 PRT Artificial Luk308 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYGERLRFGEAIDLLVPGSERSYDLTGLKP GTEYHVGISGVKGGWFSNPLRAIFTT 296 PRT Artificial Luk309 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF KIFYLESDWEGEAIALWVPGSERSYDLTGLKP GTEYFVFIHGVKGGYISIPLHANFTT 297 PRT Artificial Luk310 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYTETAKWGEAITLLVPGSERSYDLTGLK PGTEYRVGIGGVKGGGWSWPLDAIFTT 298 PRT Artificial Luk311 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WISYVEPDDGEAIELLVPGSERSYDLTGLKPG TEYIVQIDGVKGGTTSVPLNARFTT 299 PRT Artificial Luk312 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYSEYPEYGEAIDLVVPGSERSYDLTGLKP GTEYRVGITGVKGGWISKPLNATFTT 300 PRT Artificial Luk313 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYNEIGKWGEAIDLIVPGSERSYDLTGLKP GTEYAVGIDGVKGGSISEPLPASFTT 301 PRT Artificial Luk314 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYTEFNFKGEAIPLDVPGSERSYDLTGLKPG TEYFVSIHGVKGGEISPPLEALFTT 302 PRT Artificial Luk315 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WISYQEPDEIGEAIELIVPGSERSYDLTGLKPG TEYFVQIDGVKGGTWSIPLNAYFTT 303 PRT Artificial Luk317 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHEFPTWGEAIDLLVPGSERSYDLTGLKP GTEYQVRISGVKGGTTSQPLQAAAT 304 PRT Artificial Luk318 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHETVGFGEAIALLVPGSERSYDLTGLKP GTEYAVAIDGVKGGWFSHPLVAYFTT 305 PRT Artificial Luk319 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYFERFNWGEAIDLLVPGSERSYDLTGLKP GTEYQVQIDGVKGGDISIPLSARFTT 306 PRT Artificial Luk320 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYSEWEQLGEAIDLIVPGSERSYDLTGLKP GTEYQVGIAGVKGGSSSFPLGAEFTT 307 PRT Artificial Luk321 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYQEAATWGEAIDLSVPGSERSYDLTGLK PGTEYHVGIVGVKGGGVSTPLVAPFTT 308 PRT Artificial Luk322 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYYESHRGGEAIDLLVPGSERSYDLTGLKP GTEYTVGITGVKGGTISYPLHAIFTT 309 PRT Artificial Luk323 LPAPKNLVVSRVTEDSARLSWTEPDAAFDSF WIAYPEPGFQGEAISLLVPGSERSYDLTGLKP GTEYEVQIAGVKGGHVSWPLVATFTT 310 PRT Artificial Luk324 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYEEVRAEGEAIELLVPGSERSYDLTGLKP GTEYVVGIDGVKGGGFSSPLVAHFTT 311 PRT Artificial Luk326 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYYERTQQGEAIELLVPGSERSYDLTGLKP GTEYWVGIDGVKGGEVSQPLKAHFTT 312 PRT Artificial Luk327 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYLEWLYHGEAIKLYVPGSERSYDLTGLKP GTEYYVVIHGVKGGFVSTPLFATFTT 313 PRT Artificial Luk329 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYWEGIGYGEAITLLVPGSERSYDLTGLKP GTEYNVGIDGVKGGDFSTPLWARFTT 314 PRT Artificial Luk330 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHEFSTYGEAIDLLVPGSERSYDLTGLKP GTEYTVKIAGVKGGATSVPLVATFTT 315 PRT Artificial Luk331 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYFEDDERGEAIVLNVPGSERSYDLTGLKP GTEYHVIIHGVKGGQISSPLYATFTT 316 PRT Artificial Luk332 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYGEWEYPGEAIELLVPGSERSYDLTGLKP GTEYHVGIDGVKGGRVSYPLRAQFTT 317 PRT Artificial Luk333 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIHYWEGLFVGEAIVLSVPGSERSYDLTGLKP GTEYAVPIYGVKGGSISKPLYALFTT 318 PRT Artificial Luk334 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIGYHEAEGFGEAIALLVPGSERSYDLTGLKP GTEYPVGISGVKGGFVSFPLWARFTT 319 PRT Artificial Luk335 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYNEIVNHGEAIDLVVPGSERSYDLTGLKP GTEYRVSIGGVKGGHWSVPLWARFTT 320 PRT Artificial Luk336 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHEWIGPGEAISLLVPGSERSYDLTGLKP GTEYWVGIAGVKGGWSSRPLSATFTT 321 PRT Artificial Luk337 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYEEPLYFGEAIDLLVPGSERSYDLTGLKP GTEYRVHIGGVKGGRVSIPLEAEFTT 322 PRT Artificial Luk338 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYEEDNAQGEAIELLVPGSERSYDLTGLKP GTEYDVKIDGVKGGRVSTPLVARFTT 323 PRT Artificial Luk339 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYWEGQWNGEAILLDVPGSERSYDLTGLK PGTEYIVPIHGVKGGWISLPLVATFTT 324 PRT Artificial Luk340 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHESPYAGEAIDLVVPGSERSYDLTGLKP GTEYAVGIAGVKGGGYSIPLRAIFTT 325 PRT Artificial Luk342 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYSEPTIYGEAIDLLVPGSERSYDLTGLKP GTEYFVGITGVKGGWNSRPLSAIFTT 326 PRT Artificial Luk343 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYTETHWFGEAINLPVPGSERSYDLTGLKP GTEYGVIIHGVKGGYISDPLWAAFTT 327 PRT Artificial Luk344 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WISYVEPVFSGEAIELLVPGSERSYDLTGLKP GTEYIVGIGGVKGGGWSIPLEAHFTT 328 PRT Artificial Luk345 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIFYIEAKFRGEAIRLGVPGSERSYDLTGLKPG TEYFVWIHGVKGGEISDPLEAPFTT 329 PRT Artificial Luk346 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYYEIVGWGEAITLLVPGSERSYDLTGLKP GTKYVVLIDGVKGGLLSQPLHAEFAT 330 PRT Artificial Luk347 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHETTRFGEAIDLLVPGSERSYDLTGLKP GTEYVVAIQGVKGGHVSQPLRAPFTT 331 PRT Artificial Luk348 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIIYLEASFRGEAIVLTVPGSERSYDLTGLKPG TEYFVSIYGVKGGHFSPPLDAIFTT 332 PRT Artificial Luk349 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYREWIQWGEAISLLVPGSERSYDLTGLKP GTEYRVGITGVNGGVTSVPLHAKFTT 333 PRT Artificial Luk350 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYHEGLSWGEAIDLLVPGSERSYDLTGLK PGTEYTVSIDGVKGGYTSEPLRASFTT 334 PRT Artificial Luk351 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYDETLTYGEAITLLVPGSERSYDLTGLKP GTEYTVGIDGVKGGRNSVPLKATFTT 335 PRT Artificial Luk353 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYHEPRAWGEAIDLLVPGSERSYDLTGLK PGTEYLVGIGGVKGGKQSKPLVAKFTT 336 PRT Artificial Luk354 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYTEQKNHGEAIDLLVPGSERSYDLTGLK PGTEYEVNIAGVKGGGWSIPLNAWFTT 337 PRT Artificial Luk355 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF KIVYVEIHYRGEAIHLSVPGSERSYDLTGLKP GTEYHVVIHGVKGGGISLPLDAPFTT 338 PRT Artificial Luk356 LPAPKNLVVSRVTEDSARLSWTAPDATFDSF WIGYSEDQRTGEAIDLVVPGSERSYDLTGLKP GTEYRVAIAGVKGGYISQPLSANFTT 339 PRT Artificial Luk357 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYFESLLTGEAISLVVPGSERSYDLTGLKPG TEYLVPIYGVKGGFISQPLIAIFTT 340 PRT Artificial Luk358 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AINYYEYYPAGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGYISPPLSAIFTT 341 PRT Artificial Luk361 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIYYYEYYANGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGYVSDPLSAIFTT 342 PRT Artificial Luk362 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIYYLEYSFTGEAIVLTVPGSERSYDLTGLKPG TEYAVYIYGVKGGWISDPLSAIFTT 343 PRT Artificial Luk363 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIYYYEWASYGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGHISRPLSAIFTT 344 PRT Artificial Luk364 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYHEYSYRGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHSVKGGSVSSPLSAIFTT 345 PRT Artificial Luk365 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIYYYEYWYGGEAIVLTVPGSERSYDLTGLK PGTEYWVGIYGVKGGYISSPLSAIFTT 346 PRT Artificial Luk366 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYYEFNWGGEAIVLTVPGSERSYDLTGLKP GTEYWVGIYGVKGGYISYPLSAIFTT 347 PRT Artificial Luk368 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIYYFESINLGDAIVLTVPGSERSYDLTGLKPG TEYYVYIYGVKGGYISYPLSAIFTT 348 PRT Artificial Luk369 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYYEYGGGGEAIVLTVPGSERSYDLTGLKP GTEYHVGIYGVKGGYISPPLSAIFTT 349 PRT Artificial Luk370 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIYYFEYWTYGEAIVLTVPGSERSYDLTGLKP GTEYYVYIYGVKGGYISDPLSAIFTT 350 PRT Artificial Luk371 LPAPKNLVVSRVIEDSARLSWTAPDAAFDSFT IFYYEYDSGEAIVLTVPGSERSYDLTGLKPGT EYTVAIFGVKGGYISAPLSAIFTT 351 PRT Artificial Luk372 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIGYEEYANAGEAIVLTVPGSERSYDLTGLKP GTEYLVFIYGVKGGYYSYPLSAIFTT 352 PRT Artificial Luk373 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIYYFEYINLGEAIVLTVPGSERSYDLTGLKPG TEYFVYIHGVKGGFVSDPLSAIFTT 353 PRT Artificial Luk374 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIEYWEYRLAGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGAVSLPLSAIFTT 354 PRT Artificial Luk375 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIDYWEYVFLGEAIVLTVPGSERSYDLTGLKP GTEYFVSITGVKGGRYSYPLSAIFTT 355 PRT Artificial Luk376 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIPYYEYWWSGEAIVLTVPGSERSYDLTGLKP GTEYWVGIYGVKGGYISSPLSAIFTT 356 PRT Artificial Luk377 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WINYWEYYPHGEAIVLTVPGSERSYDLTGLK PGTEYFVGIYGVKGGSYSHPLSAIFTT 357 PRT Artificial Luk378 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYHEDAYTGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGFLSRPLSAIFTT 358 PRT Artificial Luk379 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YILYHEYEYSGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGLYSAPLSAIFTT 359 PRT Artificial Luk380 LPAPKNLVVSRVNEDSARLSWTAPDAAFDSF DIVYGVGEAIVLTVPGSERSYDLTGLKPGTEY YVPIAGVKGGGVSWPLSAIFTT 360 PRT Artificial Luk381 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIYYYEYYKYGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGEISDPLSAIFTT 361 PRT Artificial Luk382 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIIYDETAQYGEAIVLTVPGSERSYDLTGLKP GTEYLVPIHGVKGGTISYPLSAIFTT 362 PRT Artificial Luk390 LPAPKNLVVSRVTEDSARLSWTYIHHGFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVVWGYWNPTQYSNPLSAIFTT 363 PRT Artificial Luk394 LPAPKNLVVSRVTEDSARLSWDQYRLNFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVNWGYFLAPEISNPLSAIFTT 364 PRT Artificial Luk399 LPAPKNLVVSRVTEDSARLSWPGQTRKFNIFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVVGIFLTFGSNPLSAIFTT 365 PRT Artificial Luk409 LPAPKNLVVSRVTEDSARLSWKYTLYQHYFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVLAWWSFGSNPLSAIFTT 366 PRT Artificial Luk412 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIGYLEYPWYGEAIVLTVPGSERSYDLTGLKP GTEYFVDIYGVKGGWWSYPLSAIFTT 367 PRT Artificial Luk414 LPAPNLLVVSRVTEDSARLSWTAPDAAFDSF WIDYIETYYYGEAIVLTVPGSERSYDLTGLKP GTEYLVDIYGVKGGWYSLPLSAIFTT 368 PRT Artificial Luk415 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF EISYTEYGISGEAIVLTVPGSERSYDLTGLKPG TEYFVDIYGVKGGYLSYPLSAIFTT 369 PRT Artificial Luk417 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIDYFEYYEFGEAIVLTVPGSERSYDLTGLKP GTEYFVDIYGVKGGSWSLPLSAIFTT 370 PRT Artificial Luk420 LPAPKNLVVSRVTEDSARLSWWLGRFNFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVKQWIISEESLSNPLSAIFTT 371 PRT Artificial Luk428 LPAPKNLVVSRVTEDSARLSWGIKEETIIFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVNIIELHLWSNPLSAIFTT 372 PRT Artificial Luk438 LPAPKNLVVSRVTEDSARLSWWRKPKRWRH FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVAPDTPTPVIISNPLSAIFT T 373 PRT Artificial Luk445 LPAPKNLVVSRVTEDSARLSWEVNTKTSNKF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVGYWLTNVVLASNPLSAIF TT 374 PRT Artificial Luk447 LPAPKNLVVSRVTEDSARLSWGIDDYFVHFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVSIHFEFTTYSRSNPLSAIFT T 375 PRT Artificial Luk449 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DISYDEYPEFGEAIVLTVPGSERSYDLTGLKP GTEYLVDIIGVKGGEISLPLSAIFTT 376 PRT Artificial Luk452 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIHYAEYPDFGEAIVLTVPGSERSYDLTGLKP GTEYIVDIWGVKGGLGSWPLSAIFTT 377 PRT Artificial Luk460 LPAPKNLVVSRVTEDSARLSWIWGGESFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVGQIGFIYRPEGSNPLSAIFTT 378 PRT Artificial Luk461 LPAPKNLVVSRVTEDSARLSWLGPTATVFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVWSLLHHRFSNPLSAIFTT 379 PRT Artificial Luk462 LPAPKNLVVSRVTEDSARLSWHPIWVDFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVRGDGFEVILHSNPLSAIFTT 380 PRT Artificial Luk463 LPAPKNLVVSRVTEDSARLSWKWFKTTAFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVLHASEIHQWESSNPLSAIFTT 381 PRT Artificial Luk464 LPAPKNLVVSRVTEDSARLSWWWPVAPFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVGAINYVYFPTWSNPLSAIFTT 382 PRT Artificial Luk465 LPAPKNLVVSRVTEDSARLSWVTDPGTNFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVWSWVHSRYSNPLSAIFTT 383 PRT Artificial Luk467 LPAPKNLVVSRVTEDSARLSWPWLQYPFFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVGYLDWHIFQLASNPLSAIFT T 384 PRT Artificial Luk468 LPAPKNLVVSRVTEDSARLSWQPSHGEFANF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVQPWYRGGHIYDFSNPLSA IFTT 385 PRT Artificial Luk470 LPAPKNLVVSRVTEDSARLSWINYSDPDFFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVYHWWYRGTPVVSNPLSAIF TT 386 PRT Artificial Luk473 LPAPKNLVVSRVTEDSARLSWLEGFFPQPLFD SFLIQYLESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVPWYHHRWWFASNPLSAIFT T 387 PRT Artificial Luk475 LPAPKNLVVSRVTEDSARLSWKQHTNTHYQF DSFLIQYQESEKVGEAIVLTVPVSERSYDLTG LKPGTEYTVSIYGVRWIDNHLKFNVHSNPLS AIFTT 388 PRT Artificial Luk476 LPAPKNLVVSRVTEDSARLSWLEGFFPQPLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVPWYHHRWWFASNPLSAIF TT 389 PRT Artificial Luk478 LPAPKNLVVSRVTEDSARLSWLEGFFPQPLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVASRWHSFPVTTSNPLSAIFT T 390 PRT Artificial Luk479 LPAPKNLVVSRVTEDSARLSWLEGFFPQPLFD SFLIQYQESEKVGEAIVLTVPGSERSYGLTGL KPGTEYTVSIYGVPWYHHRWWFASNPLSAIF TT 391 PRT Artificial Luk483 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYYERFTWGEAIVLTVPGSERSYDLTGLKP GTEYPVHIWGVKGGIDSRPLSAIFTT 392 PRT Artificial Luk486 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIRYGEETVHGEAIALIVPGSERSYDLTGLKP GTEYPVAIAGVKGGTWSIPLSAIFTT 393 PRT Artificial Luk487 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIHYLEFHYAGEAIVLTVPGSERSYDLTGLKP GTEYWVVIYGVKGDLISGPLSAIFTT 394 PRT Artificial Luk498 LPAPKNLVVSRVTEDSARLSWLPGPFRRFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVNHEWYHAFSNPLSAIFTT 395 PRT Artificial Luk499 LPAPKNLVVSRVTEDSARLSWIGRELIWFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVTHEWRSEFSNPLSAIFTT 396 PRT Artificial Luk500 LPAPKNLVVSRVTEDSARLSWKKPSYYIFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVNHEWYHAFSNPLSAIFTT 397 PRT Artificial Luk505 LPAPKNLVVSRVTEDSARLSWQQAARWFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVTREWFHSFSNPLSAIFTT 398 PRT Artificial Luk507 LPAPKNLVVSRVTEDSARLSWQHHGFRLFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVFTHEWFHEFSNPLSAIFTT 399 PRT Artificial Luk510 LPAPKNLVVSRVTEDSARLSWDEYSVTTWW FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVNELYRPWVASNPLSAIF TT 400 PRT Artificial Luk513 LPAPKNLVVSRVTEDSARLSWWTGGWRRNP FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVQLHRTIIAGESNPLSAIF TT 401 PRT Artificial Luk516 LPAPKNLVVSRVTEDSARLSWVGANSRHWF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVAVSEWFHSFSNPLSAIFTT 402 PRT Artificial Luk517 LPAPKNLVVSRVTEDSARLSWVNHLEGEAW FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVQTHEWWHKFSNPLSAI FTT 403 PRT Artificial Luk519 LPAPKNLVVSRVTEDSARLSWDLEHHNYHYF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWFLQPAIHPPSNPLSAIFT T 404 PRT Artificial Luk520 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYYEYWSNGEAIVLTVPGSERSYDLTGLKP GTEYWVGIHGVKGGLISHPLSAIFTT 405 PRT Artificial Luk521 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI ITYEEATLNGEAIVLTVPGSERSYDLTGLKPG TEYTVGITGVKGGLGSYPLSAIFTT 406 PRT Artificial Luk522 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYLEQRFQGEAIVLTVPGSERSYDLTGLKP GTEYAVIIHGVKGGWISFPLSAIFTT 407 PRT Artificial Luk523 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIPYLERQLYGEAIVLTVPGSERSYDLTGLKP GTEYTVTIGGVKGGAPSRPLSAIFTT 408 PRT Artificial Luk524 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF FIFYLEYAHPGEAIVLTVPGSERSYDLTGLKP GTEYHVIIHGVKGGLISEPLSAIFTT 409 PRT Artificial Luk525 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIFYWESVTGGEAIVLTVPGSERSYDLTGLKP GTEYIVIIHGVKGGLISDPLSAIFTT 410 PRT Artificial Luk526 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYDEHHQWGEAIVLTVPGSERSYDLTGLKP GTEYWVAIYGVKGGYYSSPLSAIFTT 411 PRT Artificial Luk527 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYWEPNEVGEAIVLTVPGSERSYDLTGLKP GTEYFVEIYGVKGGEISYPLSAIFTT 412 PRT Artificial Luk528 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIDYREETPKGEAIVLTVPGSERSYDLTGLKP GTEYWVIILGVKGGGDSFPLSAIFTT 413 PRT Artificial Luk529 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIYYGEWNPKGEAIVLTVPGSERSYDLTGLKP GTEYWVIISGVKGGPQSIPLSAIFTT 414 PRT Artificial Luk530 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIFYHEIEENGEAIVLTVPGSERSYDLTGLKPG TEYFVAIHGVKGGVISTPLSAIFTT 415 PRT Artificial Luk531 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYYELYHAGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGQISLPLSAIFTT 416 PRT Artificial Luk532 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYLELESSGEAIVLTVPGSERSYDLTGLKPG TEYNVIIHGVKGGFISSPLSAIFTT 417 PRT Artificial Luk533 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYVELRNPGEAIVLTVPGSERSYDLTGLKPG TEYHVVIHGVKGGFISHPLSAIFTT 418 PRT Artificial Luk534 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYTEWNEFGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGQISVPLSAIFTT 419 PRT Artificial Luk535 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYLEPTTQGEAIVLTVPGSERSYDLTGLKPG TEYFVIIHGVKGGPVSGPLSAIFTT 420 PRT Artificial Luk536 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIAYIETDGWGEAIVLTVPGSERSYDLTGLKP GTEYFVPIHGVKGGYISQPLSAIFTT 421 PRT Artificial Luk537 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYHEHKIRGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGYISLPLSAIFTT 422 PRT Artificial Luk538 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYLERANRGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGTISDPLSAIFTT 423 PRT Artificial Luk539 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYLETLYHGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGIISSPLSAIFTT 424 PRT Artificial Luk540 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIEYPEDTEQGEAIVLTVPGSERSYDLTGLKP GTEYNVHITGVKGGSKSAPLSAIFTT 425 PRT Artificial Luk541 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NILYTETEQSGEAIVLTVPGSERSYDLTGLKP GTEYIVIIHGVKGGFISGPLSAIFTT 426 PRT Artificial Luk542 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYPEFRGDGEAIVLTVPGSERSYDLTGLKPG TEYGVIIHGVKGGGDSNPLSAIFTT 427 PRT Artificial Luk543 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYTETFHYGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGDISAPLSAIFTT 428 PRT Artificial Luk544 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYLEEFWLGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGFISVPLSAIFTT 429 PRT Artificial Luk545 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIAYIEERWSGEAIVLTVPGSERSYDLTGLKP GTEYFVLIHGVKGGFISNPLSAIFTT 430 PRT Artificial Luk546 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIYYGEWPHGGEAIVLTVPGSERSYDLTSLKP GTEYFVLIIGVKGGQLSHPLSAIFTT 431 PRT Artificial Luk547 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYLESSGTGEAIVLTVPGSERSYDLTGLKPG TEYLVIIHGVKGGRISNPLSAIFTT 432 PRT Artificial Luk548 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIYYGEWHPDGEAIVLTVPGSERSYDLTGLKP GTEYWVFILGVKGGQNSQPLSAIFTT 433 PRT Artificial Luk549 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYAESGNWGEAIVLTVPGSERSYDLTGLKP GTEYFVFIWGVKGGHESHPLSAIFTT 434 PRT Artificial Luk550 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYAETDTKGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGSISVPLSAIFTT 435 PRT Artificial Luk551 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYQEYSNHGEAIVLTVPGSERSYDLTGLKP GTEYFVPIYGVKGGFISRPLSAIFTT 436 PRT Artificial Luk552 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYDENLWLGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGFISQPLSAIFTT 437 PRT Artificial Luk553 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYAEHEKWGEAIVLTVPGSERSYDLTGLKP GTEYWVAIHGVKGGHISRPLSAIFTT 438 PRT Artificial Luk554 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIFYLETFRRGEAIVLTVPGSERSYDLTGLKPG TEYLVIIHGVKGGYVSDPLSAIFTT 439 PRT Artificial Luk555 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIFYPETNYQGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGYISDPLSAIFTT 440 PRT Artificial Luk556 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYIEEETNGEAIVLTVPGSERSYDLTGLKPG TEYQVIIHGVKGGFISLPLSAIFTT 441 PRT Artificial Luk557 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYPEVNFRGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGYISTPLSAIFTT 442 PRT Artificial Luk558 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYHEWWKSGEAIVLTVPGSERSYDLTGLK PGTEYHVVIHGVKGGHISTPLSAIFTT 443 PRT Artificial Luk559 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYPETRPTGEAIVLTVPGSERSYDLTGLKPG TEYFVVIYGVKGGWISPPLSAIFTT 444 PRT Artificial Luk560 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYIEHVQVGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGIISPPLSAIFTT 445 PRT Artificial Luk561 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYYPELYFHGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGFISPPLSAIFTT 446 PRT Artificial Luk562 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYKEYTHGGEAIVLTVPGSERSYDLTGLKP GTEYWVIIHSVKGGSISYPLSAIFTT 447 PRT Artificial Luk563 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYPEHYQDGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGWISSPLSAIFTT 448 PRT Artificial Luk564 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYIEFRYPGEAIVLTVPGSERSYDLTGLKPG TEYFVAIHGVKGGYISDPLSAIFTT 449 PRT Artificial Luk565 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYLETWGSGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGLISSPLSAIFTT 450 PRT Artificial Luk566 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYYEHADAGEAIVLTVPGSERSYDLTGLKP GTEYFVIIHGVKGGYISKPLSAIFTT 451 PRT Artificial Luk567 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYQEDSDHGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGTISKPLSAIFTT 452 PRT Artificial Luk568 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYIEEHDVGEAIVLTVPGSERSYDLTGLKPG TEYIVIIHGVKGGYISDPLSAIFTT 453 PRT Artificial Luk569 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYPETQTIGEAIVLTVPGSERSYDLTGLKPG TEYFVGIHGVKGGIISDPLSAIFTT 454 PRT Artificial Luk570 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IYYGEWREHGEAIVLTVPGSERSYDLTGLKPG TEYFVLIQGVKGGQTSGPLSAIFTT 455 PRT Artificial Luk571 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF KIFYLEPKENGEAIVLTVPGSERSYDLTGLKP GTEYFVIITGVKGGFISEPLSAIFTT 456 PRT Artificial Luk572 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIHYDEWENGGEAIVLTVPGSERSYDLTGLKP GTEYWVIIIGVKGGVRSNPLSAIFTT 457 PRT Artificial Luk575 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PIFYVEIPQPGEAIVLTVPGSERSYDLTGLKPG TEYFVIIHGVKGGGISDPLSAIFTT 458 PRT Artificial Luk576 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VILYHEYWASGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGFLSDPLSAIFTT 459 PRT Artificial Luk577 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYCEHWTSGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGEISAPLSAIFTT 460 PRT Artificial Luk578 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYQEHLGYGEAIVLTVPGSERSYDLTGLKP GTEYVVVIHGVKGGWISSPLSAIFTT 461 PRT Artificial Luk579 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYEETANGGEAIVLTVPGSERSYDLTGLKPG TEYFVVIHGVKGGHISSPLSAIFTT 462 PRT Artificial Luk580 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYPETQKYGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGFISSPLSAIFTT 463 PRT Artificial Luk581 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYGEYENGGEAIVLTVPGSERSYDLTGLKP GTEYFVIIVGVKGGFDSKPLSAIFTT 464 PRT Artificial Luk582 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYHETVDKGEAIVLTVPGSERSYDLTGLKP GTEYFVVVSGVKGGYISDPLSAIFTT 465 PRT Artificial Luk583 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYREESKYGEAIVLTVPGSERSYDLTGLKP GTEYFVPIHGVKGGEISDPLSAIFTT 466 PRT Artificial Luk584 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYQEVVEWGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGWISDPLSAIFTT 467 PRT Artificial Luk585 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYPETWIAGEAIVLTVPGSERSYDLTGLKPG TEYLVVIHGVKGGIISWPLSAIFTT 468 PRT Artificial Luk587 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIPYQEYLGWGEAIVLTVPGSERSYDLTGLKP GTEYWVGIYGVKGGFISPPLSAIFTT 469 PRT Artificial Luk588 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYYEHQVAGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGWISSPLSAIFTT 470 PRT Artificial Luk589 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYAEEQRNGEAIVLTVPGSERSYDLTGLKPG TEYFVIIHGVKGGFISPPLSAIFTT 471 PRT Artificial Luk590 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIFYLEERLTGEAIVLTVPGSERSYDLTGLKPG TEYLVVIHGVKGGVISDPLSAIFTT 472 PRT Artificial Luk592 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYYEAVHQGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGNISVPLSAIFTT 473 PRT Artificial Luk593 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYVELVWKGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGYISDPLSAIFTT 474 PRT Artificial Luk594 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIHYGEYENGGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGFISDPLSAIFTT 475 PRT Artificial Luk595 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIIYFETKAYGEAIVLTVPGSERSYDLTGLKPG TEYWVIIHGVKGGYISVPLSAIFTT 476 PRT Artificial Luk596 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYDEEWSKGEAIVLTVPGSERSYDLTGLKP GTEYAVFIYGVKGGAISEPLSAIFTT 477 PRT Artificial Luk597 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIHYLETDPGGEAIVLTVPGSERSYDLTGLKP GTEYFVSIYGVKGGWISPPLSAIFTT 478 PRT Artificial Luk598 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYDEDRPQGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGYLSIPLSAIFTT 479 PRT Artificial Luk599 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIFYREETPHGEAIVLTVPGSERSYDLTGLKP GTEYWVLILGVKGGGISEPLSAIFTT 480 PRT Artificial Luk601 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYIEDNKVGEAIVLTVPGSVRSYDLTGLKP GTEYFVVIHGVKGGIISEPLSAIFTT 481 PRT Artificial Luk602 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYQELNRDGEAIVLTVPGSERSYDLTGLKP GTEYLVIIHGVKGGFISPPLSAIFTT 482 PRT Artificial Luk603 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYLEFWYRGEAIVLTVPGSERSYDLTGLKP GTEYNVIIHGVKGGWISEPLSAIFTT 483 PRT Artificial Luk604 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYGEWPQEGEAIVLTVPGSERSYDLTGLKP GTEYWVVILGVKGGQASPPLSAIFTT 484 PRT Artificial Luk605 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIHYLEHAARGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGYISFPLSAIFTT 485 PRT Artificial Luk606 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYLESYPRGEAIVLTVPGSERSYDLTGLKP GTEYFVAIYGVKGGYLSPPLSAIFTT 486 PRT Artificial Luk607 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYHEWVPWGEAIVLTVPGSERSYDLTGLK PGTEYFVVIHGVKGGTISFPLSAIFTT 487 PRT Artificial Luk608 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYGEYENGGEAIVLTVPGSERSYDLTGLKP GTEYFVFIIGVKGGPDSLPLSAIFTT 488 PRT Artificial Luk609 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF DIRYWEGPGYGEAIVLTVPGSERSYDLTGLKP GTEYRVRIVGVKGGKRSEPLSAIFTT 489 PRT Artificial Luk610 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF FIYYGEYDPVGEAIVLTVPGSERSYDLTGLKP GTEYFVIIQGVKGGQASGPLSAIFTT 490 PRT Artificial Luk611 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIAYAEFWRYGEAIVLTVPGSERSYDLTGLKP GTEYWVNIAGVKGGEWSKPLSAIFTT 491 PRT Artificial Luk612 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYQEESKYGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGAISQPLSAIFTT 492 PRT Artificial Luk613 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF NIFYIETDKPGEAIVLTVPGSERSYDLTGLKPG TEYFVAIHGVKGGFISEPLSAIFTT 493 PRT Artificial Luk614 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIFYTEPVGHGEAIVLTVPGSERSYDLTGLKP GTEYFVAIHGVKGGTISPPLSAIFTT 494 PRT Artificial Luk615 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYIEHRLQGEAIVLTVPGSERSYDLTGLKPG TEYLVLIHGVKGGFISPPLSAIFTT 495 PRT Artificial Luk616 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIFYHEGLKSGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGTISNPLSAIFTT 496 PRT Artificial Luk617 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYHETRVTGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGYISEPLSAIFTT 497 PRT Artificial Luk618 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIFYQEAVEGGEAIVLTVPGSERSYDLTGLKP GTEYFVPIHGVKGGWISQPLSAIFTT 498 PRT Artificial Luk619 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QILYVEEFTRGEAIVLTVPGSERSYDLTGLKP GTEYVVIIHGVKGGYISKPLSAIFTT 499 PRT Artificial Luk620 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TYIEALGFGEAIVLTVPGSERSYDLTGLKPGT EYFVAIYGVKGGYISEPLSAIFTT 500 PRT Artificial Luk621 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIFYLEHWNPGEAIVLTVPGSERSYDLTGLKP GTEYLVPIHGVKGGSISPPLSAIFTT 501 PRT Artificial Luk622 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIFYVEWEVVGEAIVLTVPGSERSYDLTGLKP GTEYFVVIHGVKGGVISNPLSAIFTT 502 PRT Artificial Luk623 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYLEESKNGEAIVLTVPGSERSYDLTGLKP GTEYQVVIHGVKGGVISPPLSAIFTT 503 PRT Artificial Luk624 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYNEDHKSGEAIVLTVPGSERSYDLTGLKP GTEYLVVIHGVKGGYISKPLSAIFTT 504 PRT Artificial Luk625 LPAPKNLVVSRVPEDSARLSWTAPDAAFDSF LIDYQEWHEGEAIHLLVPGSERSYDLTGLKPG TEYAVIIVGVKGGKGSHPLSAIFTT 505 PRT Artificial Luk626 LPAPKNLVVSRVTEDSARLSWTAPYAAFDSF WIGYYETTIPGEAIDLVVPGSERSYDLTGLKP GTEYGVGIDGVKGGRYSHPLSAIFTT 506 PRT Artificial Luk627 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIFYKEEAIPGEAIALIVPGSERSYDLTGLKPG TEYFVPIHGVKGGYISTPLSAIFTT 507 PRT Artificial Luk628 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIDYTELHNWGEAIHLFVPGSERSYDLTGLKP GTEYTVLIVGVKGGTGSIPLSAIFTT 508 PRT Artificial Luk629 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YITYEEEWWTGEAIFLDVPGSERSYDLTGLKP GTEYLVTIKGVKGGPWSQPLSAIFTT 509 PRT Artificial Luk630 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF AIWYHEWGPVGEAILLYVPGSERSYDLTGLK PGTEYPVAIHGVKGGGTSHPLSAIFTT 510 PRT Artificial Luk631 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYEELRYKGEAIWLFVPGSERSYDLTGLKP GTEYHVHIWGVKGGYFSRPLSAIFTT 511 PRT Artificial Luk632 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IDYWEQWNTGEAIHLYVPGSERSYDLTGLKP GTEYSVYIVGVKGGYASWPLSAIFTT 512 PRT Artificial Luk633 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYDENHLYGEAIDLVVPGSERSYDLTGLK PGTEYTVSIAGVKGGLESFPLSAIFTT 513 PRT Artificial Luk634 LPAPKNLVVSRVTEDSARLSWTAPEAAFDSF HISYWEFPLGGEAIGLWVPGSERSYDLTGLKP GTEYFVIIAGVKGGEFSNPLSAIFTT 514 PRT Artificial Luk635 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFI IEYHEWFAKGEAIGLVVPGSERSYDLTGLKPG TEYSVIIVGVKGGAYSFPLSAIFTT 515 PRT Artificial Luk636 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LIDYWEGEFDGEAIHLFVPGSERSYDLTGLKP GTEYDVFIVGVKGGHGSDPLSAIFTT 516 PRT Artificial Luk637 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF QIPYYELDSVGEAIVLTVPGSERSYDLTGLKP GTEYFVGIYGVKGGYISPPLSAIFTT 517 PRT Artificial Luk638 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIGYGEYDPTGEAIVLTVPGSERSYDLTGLKP GTEYWVLISGVKGGYYSDPLSAIFTT 518 PRT Artificial Luk639 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIYYLESVARGEAIVLTVPGSERSYDLTGLKP GTEYFVPIYGVKGGYISYPLSAIFTT 519 PRT Artificial Luk640 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIPYWESYYSGEAIVLTVPGSERSYDLTGLKP GTEYVVAIYGVKGGYISSPLSAIFTT 520 PRT Artificial Luk641 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIYYGEYHSGGEAIVLTVPGSERSYDLTGLKP GTEYFVLIDGVKGGLYSGPLSAIFTT 521 PRT Artificial Luk642 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIVYAEYYWYGEAIVLTVPGSERSYDLTGLK PGTEYYVYIAGVKGGYGSDPLSAIFTT 522 PRT Artificial Luk643 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIPYYESNLGGEAIVLTVPGSERSYDLTGLKP GTEYWVGIYGVKGGHISSPLSAIFTT 523 PRT Artificial Luk644 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TILYYELIDSGEAIVLTVPGSERSYDLTGLKPG TEYFVGIYGVKGGYISLPLSAIFTT 524 PRT Artificial Luk645 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WISYGEYWPSGEAIVLTVPGSERSYDLTGLKP GTEYFVLIRGVKGGDYSNPLSAIFTT 525 PRT Artificial Luk646 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF RIFYGEYDGGGEAIVLTVPGSERSYDLTGLKP GTEYGVYIYGVKGGYISQPLSAIFTT 526 PRT Artificial Luk647 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SIYYGEWDPTGEAIVLTVPGSERSYDLTGLKP GTEYWVLIVGVKGGSTSAPLSAIFTT 527 PRT Artificial Luk648 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIGYEEYYLVGEAIVLTVPGSERSYDLTGLKP GTEYLVWIKGVKGGYVGRPLSAIFTT 528 PRT Artificial Luk649 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIAYSERVRYGEAIVLTVPGSERSYDLTGLKP GTEYWVGISGVKGGPYSEPLSAIFTT 529 PRT Artificial Luk650 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF TIHYYESYTVGEAIVLTVPGSERSYDLTGLKP GTEYWVGIYGVKGGYISEPLSAIFTT 530 PRT Artificial Luk651 LPAPKNLVLSRVTEDSARLSWAQATYYQFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVLEVIPKLRYKVYSNPLSAIFT T 531 PRT Artificial Luk652 LPAPKNLVVSRVTEDSARLSWSEVEDIPFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVLHYNRGQHPWHSNPLSAIFTT 532 PRT Artificial Luk653 LPAPKNLVVSRVTEDSARLSWNLEVAFYFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVEHLDEVWWTANLSNPLSAIF TT 533 PRT Artificial Luk654 LPAPKNLVVSRVTEDSARLSWSHFPNDWFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVHYWQFDIQSNPLSAIFTT 534 PRT Artificial Luk655 LPAPKNLVVCRVTEDSARLSWRTYTSDAGFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVHEHAHIQYWHWSNPLSAI FTT 535 PRT Artificial Luk656 LPAPKNLVVSRVTEDSARLSWKREQWANYF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWEHLYKELWSYTSNPLS AIFTT 536 PRT Artificial Luk657 LPAPKNLVVSRVTEDSARLSWSELEARTHFD SFLIQYQESEKVSEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVAQQLVAWRGSNPLSAIFTT 537 PRT Artificial Luk658 LPAPKNLVVSRVTEDSARLSWKRARLDLPFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVDAWIPTVGSNPLSAIFTT 538 PRT Artificial Luk659 LPAPKNLVVSRVTEDSARLSWINYWVLNYFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVEVWPQDHEWIDSNPLSAIF TT 539 PRT Artificial Luk660 LPAPKNLVVSRVTEDSARLSWYREVDFTTFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVSSYYILHSNPLSAIFTT 540 PRT Artificial Luk661 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF LITYREQIFAGEVIVLTVPGSERSYDLTGLKPG TEYPVCIYGVKGGPISDPLSAIFTT 541 PRT Artificial Luk662 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VINYREVINEGEAIILHVPGSERSYRPERSETG YRIHRHHSWC 542 PRT Artificial Luk663 LPAPKNLVVSRVTEDSARLSWVVHNHLAFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVEPYVYAQYESNPLSAIFTT 543 PRT Artificial Luk664 LPAPKNLVVSRVTEDSARLSWKRKSGAPFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVHGWDPGSDSNPLSAIFTT 544 PRT Artificial Luk665 LPAPKNLVVSRVTEDSARLSWWHVRGHDFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVVWLPTTDSNPLSAIFTT 545 PRT Artificial Luk666 LPAPKNLVVSRVTEDSARLSWSPDRARFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVWSWDNDDASNPLSAIFTT 546 PRT Artificial Luk667 LPAPKNLVVSRVTEDSARLSWFAGLQLFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVDWTVEEQSYHLWSNPLSAIFT T 547 PRT Artificial Luk668 LPAPKNLVVSRVTEDSARLSWTIPHYTFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGT EYTVSIYGVPGKYIEPRWHFSNPLSAIFTT 548 PRT Artificial Luk669 LPAPKNLVVSRVTEDSARLSWKRYSWLFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVLTWDPNDPSNPLSAIFTT 549 PRT Artificial Luk670 LPAPKNLVVSRVTEDSARLSWKTIVTTIFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVDHRGFPFWQYWSSNPLSAIFT T 550 PRT Artificial Luk671 LPAPKNLVVSRVTEDSARLSWYARRIYFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVETPNPYYDSNPLSAIFTT 551 PRT Artificial Luk672 LPAPKNLVVSRVTEDSARLSWNLEQSTFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVTTYRITVPVRDHSNPLSAIFTT 552 PRT Artificial Luk673 LPAPKNLVVSRVTEDSARLSWRAAGTGFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVNWQPDYWTWPSNPLSAIFTT 553 PRT Artificial Luk674 LPAPKNLVVSRVTEDSARLSWPISHLSFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGT EYTVSIYGVWHQTVGRWFSNPLSAIFTT 554 PRT Artificial Luk675 LPAPKNLVVSRVTEDSARLSWVRKKVNRFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVRNWKPNDPSNPLSAIFTT 555 PRT Artificial Luk676 LPAPKNLVVSRVTEDSARLSWVSATQHPFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVDNWDPTDPSNPLSAIFTT 556 PRT Artificial Luk677 LPAPKNLVVSRVTEDSARLSWPIALRDFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVISWDPTDPSNPLSAIFTT 557 PRT Artificial Luk678 LPAPKNLVVSRVTEDSARLSWWDAEWFAPH FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVGLLKWPNYAVLSNPLS AIFTT 558 PRT Artificial Luk679 LPAPKNLVVSRVTEDSARLSWPNNQRYYQPF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVLSWNPHHWSNPLSAIFTT 559 PRT Artificial Luk680 LPAPKNLVVSRVTEDSARLSWYDARVTDEFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVDDLLTNHLLAISNPLSAIF TT 560 PRT Artificial Luk681 LPAPKNLVVSRVTEDSARLSWKKRNTLKIFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVETWDPNDWSNPLSAIFTT 561 PRT Artificial Luk682 LPAPKNLVVSRVTEDSARLSWLNRVKPNDFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVITWHPGHWSNPLSAIFTT 562 PRT Artificial Luk683 LPAPKNLVVSRVTEDSARLSWLTVRFTKFEF DSFLIQYQESEKVGEAIVLIVPGSERSYDLTGL KPGTEYTVSIYGVRSSKPRASNPLSAIFTT 563 PRT Artificial Luk684 LPAPKNLVVSRVTEDSARLSWPNYRKVVSVF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVHTWHPGHYSNPLSAIFTT 564 PRT Artificial Luk685 LPAPKNLVVSRVTEDSARLSWGNRQQVRSAF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVVGWHPNHPSNPLSAIFTT 565 PRT Artificial Luk686 LPARKTWLFLVLPKTLRVCLGPRRTRRSTLFG LRTQRLLSGGKRLACWCRVLNVLTT 566 PRT Artificial Luk687 LPARKTWLFLVLPKTLRVCLGPRRTRRSTLSG LHTQRRHPGVKRSA 567 PRT Artificial Luk688 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIEGGEYYYVGEAIVLTVPGSERSYDLTGLKP GTEYGVPIGGVKGGPNSHPLSAIFTT 568 PRT Artificial Luk689 LPAPKNLVVSRVTEDSARLSWWFYLTSWFAF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVLKVDPHVRSNPLSAIFTT 569 PRT Artificial Luk690 LPAPKNLVVSRVTEDSARLSWYHVNFGFFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVYEDYPVIIFNNRSNPLSAIFT T 570 PRT Artificial Luk691 LPAPKNLVVSRVTEDSARLSWEDIKNKRFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVRGLANPFRVSNPLSAIFTT 571 PRT Artificial Luk692 LPAPKNLVVSRVTEDSARLSWWRYGPWFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVRTHVRPPQWVSNPLSAIF TT 572 PRT Artificial Luk693 LPAPKAAVVSRVTEDSARLSWWRYGPWFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVRTHVRPPQWVSNPLSAIF TT 573 PRT Artificial Luk694 LPAPKNLGCFSCYRRLCPSVLD 574 PRT Artificial Luk695 LPAPKNLVVSRVTEDSARLSWLNWEQYITFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVWIIRDRSHWLNPSNPLSAIF TT 575 PRT Artificial Luk696 LPAPKNLWLFLVLPKTLPVCLGGVMARGSTS TLS 576 PRT Artificial Luk697 LPAPKNLVVSRVTEDSARLSWERFGPWFHFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVKTQPEQEFKSNPLSAIFTT 577 PRT Artificial Luk698 LPAPKNPGCFSCYRRLCPSVLVALWPVVPLR LFPDPVPGIRKSW 578 PRT Artificial Luk699 LPAPKNLVVSRVTEDSARLSWWRYGPWFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVKQHHSLFHSNPLSAIFTT 579 PRT Artificial Luk700 LPAPKNLVVSRVTEDSARLSWNQQLNYQYF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWYRWWSGSNPLSAIFTT 580 PRT Artificial Luk701 LPAPKNLVVSRVTEDSARLSWWRYGPWFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVRTHNNQPGHYTSNPLSAI FTT 581 PRT Artificial Luk702 LPAPTKNGCFSCYRRLCPSVLVALWPVVPLR LFPDPVPGIRKSW 582 PRT Artificial Luk703 LPAPKALVVSRVTEDSARLSWWRYGPWFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVRTHVRPPQWVSNPLSAIF TT 583 PRT Artificial Luk704 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PILYQERWWKGEAIVLTVPGSERSYDLTGLK PGTEYGVPITGVKGGGVSFPLSAIFTT 584 PRT Artificial Luk705 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HIWYRESWYFGEAIVLTVPGSERSYDLTGLKP GTEYYVVIRGVKGGSLSWPLSAIFTT 585 PRT Artificial Luk706 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF PISYYEQPRRGEAIWLFVPGSERSYDLTGLKP GTEYTVYITGVKGGTWSFPLTATFTT 586 PRT Artificial Luk707 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF GIEYYETKTKGEAINLFVPGSERSYDLTGLKP GTEYYVIILGVKGGEPSSPLVAPFTT 587 PRT Artificial Luk708 LPAPKNLVVSRVTEDSARLSWKDVGEWKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVHQLTITYSPTSNPLSAIFTT 588 PRT Artificial Luk709 LPAPKNLVVSRVTEDSARLSWKRSYHPNFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVIDVPTVYHPGRSNPLSAIFTT 589 PRT Artificial Luk710 LPAPKNLVVSRVTEDSARLSWLKKVSKFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVGEFPDRIYWGASNPLSAIFTT 590 PRT Artificial Luk711 LPAPKNLVVSRVTEDSARLSWYYWVQTIFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVGNLPDIFYKLPSNPLSAIFTT 591 PRT Artificial Luk712 LPAPKNLVVSRVTEDSARLSWSKKLENFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVYHTHLIFSNPLSAIFTT 592 PRT Artificial Luk713 LPAPKNLVVSRVTEDSARLSWHDLTIWPFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVVIEFEAWSNPLSAIFTT 593 PRT Artificial Luk714 LPAPKNLVVSRVTEDSARLSWFPWTEWSAFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVENWLVLATATWSNPLSAIF TT 594 PRT Artificial Luk715 LPAPKNLVVSRVTEDSARLSWVEWWIRPIEF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWQQLYVEILISNPLSAIFT T 595 PRT Artificial Luk716 LPAPKNLVVSRVTEDSARLSWSSQRTLPREFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVHVIIHSGSSNPLSAIFTT 596 PRT Artificial Luk717 LPAPKNLVVSRVTEDSARLSWTSRLEDFWFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVGSEVYFRYYEHWSNPLSAI FTT 597 PRT Artificial Luk718 LPAPKNLVVSRVTEDSARLSWQVNRNAQFHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVAHPKLVWFAPPSNPLSAI FTT 598 PRT Artificial Luk719 LPAPKNLVVSRVTEDSARLSWTFLEKWFIFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVKHHDHDPEYPSNPLSAIFT T 599 PRT Artificial Luk720 LPAPKNLVVSRVTEDSARLSWRHPRIQGGHF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVIHPFWWSPSNPLSAIFTT 600 PRT Artificial Luk721 LPAPKNLVVSRVTEDSARLSWYNAKKITPFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVYPEVQHTTSNPLSAIFTT 601 PRT Artificial Luk722 LPAPKNLVVSRVTEDSARLSWTEPWQEFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVILPTLHKSNPLSAIFTT 602 PRT Artificial Luk723 LPAPKNLVVSRVTEDSARLSWYRFPRIHFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVHTNIDLHNYNYLSNPLSAIFT T 603 PRT Artificial Luk724 LPAPKNLVVSRVTEDSARLSWAERHPWFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVLRQNINIQDTNYSNPLSAIFTT 604 PRT Artificial Luk725 LPAPKNLVVSRVTEDSARLSWPWWEGWTFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVKIRTLKASRSNPLSAIFTT 605 PRT Artificial Luk726 LPAPKNLVVSRVTEDSARLSWAANFIDFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVYSPKLRWDLLNYSNPLSAIFTT 606 PRT Artificial Luk727 LPAPKNLVVSRVTEDSARLSWFKQEFEFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPG TEYTVSIYGVYYPEYYPREPWPSNPLSAIFTT 607 PRT Artificial Luk728 LPAPKNLVVSRVTEDSARLSWEDEGTQFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVHWYWYWQRSNPLSAIFTT 608 PRT Artificial Luk729 LPAPKNLVVSRVTEDSARLSWFGNQTGARSF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVYYQFRRTVRNSNPLSAIF TT 609 PRT Artificial Luk730 LPAPKNLVVSRVTEDSARLSWGENRFVLSFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVLYHARHTWWLQQSNPLSA IFTT 610 PRT Artificial Luk731 LPAPKNLVVSRVTEDSARLSWEKQQLKKWSF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTGYTVSIYGVEHSNTRKRHSNPLSAIFT T 611 PRT Artificial Luk732 LPAPKNLVVSRVTEDSARLSWKINDNSGYFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVAHRYENNPTLWSNPLSAIF TT 612 PRT Artificial Luk733 LPAPKNLVVSRVTEDSARLSWPAFRWQPPGF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVGGFLYPWNYPTSNPLSAI FTT 613 PRT Artificial Luk734 LPAPKNLVVSRVTEDSARLSWISEKPTTSLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVWPRAIHYAYWFNSNPLSAI FTT 614 PRT Artificial Luk735 LPAPKNLVVSRVTEDSARLSWQKSFQLTPFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVVEYKWAATNPSNPLSAIFT T 615 PRT Artificial Luk736 LPAPKNLVVSRVTEDSARLSWNASLNANHFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVVTSESNYGSNPLSAIFTT 616 PRT Artificial Luk737 LPAPKNLVVSRVTEDSARLSWTNTARLNKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVAITHSHHHHSSNPLSAIFTT 617 PRT Artificial Luk738 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF HILYWEPTPIGEAILLNVPGSERSYDLTGLKPG TEYNVEIDGVKGGNPSDPLSAIFTT 618 PRT Artificial Luk739 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF SISYREGGQHGEAIVLTVPGSERSYDLTGLKP GTEYSVYILGVKGGDESEPLSAIFTT 619 PRT Artificial Luk740 LPAPKNLVVSRVTEDSARLSWPWWNKHFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVIQWKKKPFSNPLSAIFTT 620 PRT Artificial Luk741 LPAPKNLVVSRVTEDSARLSWPWWNKHFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVGEHDWYLLNYAESNPLSAIF TT 621 PRT Artificial Luk742 LPAPKNLVVSRVTEDSARLSWWAFSYLQFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVEVRENSWNHSSNPLSAIFT T 622 PRT Artificial Luk743 LPAPKNLVVSRVTEDSARLSWRETHNPQFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVIQWKKKPFSNPLSAIFTT 623 PRT Artificial Luk744 LPAPKNLVVSRVTEDSARLSWTTRVDEFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVKWYNWKKNVNTESNPLSAIF TT 624 PRT Artificial Luk745 LPAPKNLVVSRVTEDSARLSWSQKDINFFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVLWYNWKKNWDNSNPLSAIFT T 625 PRT Artificial Luk746 LPAPKNLVVSRVTEDSARLSWFTTNNHWFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVIQWKKKPFSNPLSAIFTT 626 PRT Artificial Luk747 LPAPKNLVVSRVTEDSARLSWGRAREPASFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVLTVLFIDSSNPLSAIFTT 627 PRT Artificial Luk748 LPAPKNLVVSRVTEDSVRLSWYNWKKKRLK FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVPNLWDIWNWVLSNPLS AIFTT 628 PRT Artificial Luk749 LPAPKNLVVSRVTEDSARLSWGTFNLEVYIFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVVSANWHGHSNPLSAIFTT 629 PRT Artificial Luk750 LPAPKNLVVSRVTEDSARLSWPQIFNELWEF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWWYNRKKKRQSNPLSAI FTT 630 PRT Artificial Luk751 LPAPKNLVVSRVTEDSARLSWYNEQKKPINF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVWWYNRKKKRQSNPLSAI FTT 631 PRT Artificial Luk752 LPAPKNLVVSRVTEDSARLSWRGKYSVVDFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVPWYNWKKKYVISNPLSAIF TT 632 PRT Artificial Luk753 LPAPKNLVVSRVTEDSARLSWYNTKKNPVFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVWLIKSLNAWFSNPLSAIFT T 633 PRT Artificial Luk754 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF VIIYEEVQWRGEAIRLFVPGSERSYDLTGLKP GTEYDVNIRGVKGGGSSAPLSAIFTT 634 PRT Artificial Luk755 LPAPKNLVVSRVTEDSARLSWYNWKKKPGY FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVVHYHEWLASNPLSAIFT T 635 PRT Artificial Luk756 LPAPKNLVVSRVTEDSARLSWYTVKKKPQKF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVLDAYPIAEWPAQSNPLSA IFTT 636 PRT Artificial Luk757 LPAPKNLVVSRVTEDSARLSWYNTKKKPQFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVNEFILRWEGSNPLSAIFTT 637 PRT Artificial Luk758 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF YIGYYELIGAGEAIVLTVPGSERSYDLTGLKP GTEYGVGIQGVKGGSYSAPLSAIFTT 638 PRT Artificial Luk759 LPAPKNLVVSRVTEDSARLSWYDRKVEFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVGVDGWGYLLLVSNPLSAIFTT 639 PRT Artificial Luk760 LPAPKNLVVSRVTEDSARLSWIVPRTFHFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVWSQYITHWLPKSNPLSAIFTT 640 PRT Artificial Luk761 LPAPKNLVVSRVTEDSARLSWNYRVATFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVSVELLSSNPLSAIFTT 641 PRT Artificial Luk762 LPAPKNLVVSRVTEDSARLSWQPHRYEFYQF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVADFEFELHSNPLSAIFTT 642 PRT Artificial Luk763 LPAPKNLVVSRVTEDSARLSWIPSYHLFAFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVNDAEQRYHHSNPLSAIFTT 643 PRT Artificial Luk764 LPAPKNLVVSRVTEDSARLSWPINKTTSPFDS FLIQYQESEKVGEAIVLTVPGSERSYDLTGLK PGTEYTVSIYGVVEAHYDAFISNPLSAIFTT 644 PRT Artificial Luk765 LPAPKNLVVSRVTEDSARLSWRKKLWEAEFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVILPDSFHVHHCNPLSAIFTT 645 PRT Artificial Luk766 LPAPKNLVVSRVTEDSARLSWKRPQWRRLFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVGVNWPASVSSNPLSAIFTT 646 PRT Artificial Luk767 LPAPKNLVVSRVTEDSARLSWIWDAIGPHFFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVSWFIRITASNPLSAIFTT 647 PRT Artificial Luk768 LPAPKNLVVSRVTEDSARLSWRGLEPRWGFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVPWYEHLRILNATSNPLSAIF TT 648 PRT Artificial Luk769 LPAPKNLVVSRVTEDSARLSWDWWSNPIKFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVINWHWYQTHRTSNPLSAIF TT 649 PRT Artificial Luk770 LPAPKNLVVSRVTEDSARLSWEQWHAGVNP FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVSYYVRVLQFALFSNPLS AIFTT 650 PRT Artificial Luk771 LPAPKNLVVSRVTEDSARLSWAQVETQIHFD SFLIQYQESEKVGESDLLTVPGSERSYDLTGL KPGTEYTVSIYGVSHYRRHVPRHSNPLSAIFT T 651 PRT Artificial Luk772 LPAPKNLVVSRVTEDSARLSWIAYYYGQTFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVWHENYAKWPDPSNPLSAI FTT 652 PRT Artificial Luk773 LPAPKNLVVSRVTEDSARLSWWHWLTHHFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVFFRWQDPLHDLISNPLSAI FTT 653 PRT Artificial Luk774 LPAPKNLVVSRVTEDSARLSWKYKEHFQIFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVERIWWQYRSNPLSAIFTT 654 PRT Artificial Luk775 LPAPKNLVVSRVTEDSARLSWVGDAYFNHLF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVEARPKPRLSNPLSAIFTT 655 PRT Artificial Luk776 LPAPKNLVVSRVTEDSARLSWNKRVPNFDSF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKP GTEYTVSIYGVIQWKKKPFSNPLSAIFTT 656 PRT Artificial Luk777 LPAPKNLVVSRVTEDSARLSWYNEQKKRSFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVPIRRSGWDVRSNPLSAIFT T 657 PRT Artificial Luk778 LPAPKNLVVSRVTEDSARLSWYNTKKKPVFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVKVDDWPDYWQSNPLSAIF TT 658 PRT Artificial Luk779 LPAPKNLVVSRVTEDSARLSWYNVKKTFQFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVAAVWYTPNTQSNPLSAIFT T 659 PRT Artificial Luk780 LPAPKNLVVSRVTEDSARLSWYNSKKKVQFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGVIESHWWQLKWPSNPLSAIF TT 660 PRT Artificial Luk781 LPAPKNLVVSRVTEDSARLSWYNTKKKTAFF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVDEVGYLHIETSNPLSAIFT T 661 PRT Artificial Luk782 LPAPKNLVVSRVTEDSARLSWYNEKKIFQFD SFLIQYQESEKVGEAIVLTVPGSERSYDLTGL KPGTEYTVSIYGAKGPNFPSQNDPSSNPLSAIF TT 662 PRT Artificial Luk783 LPAPKNLVVSRVTEDSARLSWYNWKKKRLK FDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVASPVYTGLYLGSNPLSA IFTT 663 PRT Artificial Luk784 LPAPKNLVVSRVTEDSARLSWYTVKKKPQKF DSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGVGDQLLEIGRTGSNPLSAIF TT 664 PRT Artificial Luk785 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF FIGYREVPFLGEAIVLTVPGSERSYDLTGLKPG TEYIVLIWGVKGGIPSQPLSAIFTT 665 PRT Artificial Luk786 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF FIPYREEAPLGEAIVLTVPGSERSYDLTGLKPG TEYDVIIVGVKGGYPSKPLSAIFTT 666 PRT Artificial Luk787 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIVYHELVHSGEAIVLTVPGSERSYDLTGLKP GTEYPVFIVGVKGGWYSPPLSAIFTT 667 PRT Artificial TCL24 LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFXIXYXEXXXXGEAIXLXVPGSERSYD LTGLKPGTEYXVXIXGVKGGXXSXPLXA XFTT 

1. A binding molecule comprising: a first binding domain capable of binding to a glycosylated staphylococcal surface protein and a second binding domain capable of binding to a staphylococcal leukotoxin.
 2. The binding molecule of claim 1, wherein the glycosylated staphylococcal surface protein is a glycosylated serine-aspartate dipeptide repeat (SDR) containing protein.
 3. The binding molecule of claim 2, wherein the glycosylated SDR-containing protein is ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG or SdrH.
 4. The binding molecule of claim 1, wherein the first binding domain is a full-length antibody or antibody fragment.
 5. The binding molecule of claim 4, wherein the full-length antibody or antibody fragment is resistant to proteolytic degradation by a staphylococcal protease that cleaves wild-type IgG1.
 6. (canceled)
 7. (canceled)
 8. The binding molecule of claim 4, wherein the full-length antibody or antibody fragment is not capable of specific binding to human FcγRI.
 9. (canceled)
 10. The binding molecule of claim 4, wherein the full-length antibody or antibody fragment is not capable of specific binding to Protein A.
 11. The binding molecule of claim 4, wherein the full-length antibody or antibody fragment is not capable of specific binding to Sbi.
 12. The binding molecule of claim 4, wherein the full-length antibody or antibody fragment is capable of specific binding to FcRn.
 13. The binding molecule of claim 1, wherein the second binding domain is an alternative scaffold.
 14. The binding molecule of claim 13, wherein the alternative scaffold comprises a fibronectin type III (FN3) domain.
 15. The binding molecule of claim 14, wherein the FN3 domain binds to LukA having the amino acid sequence of SEQ ID NO:10, LukB having the amino acid sequence of SEQ ID NO:11, LukD having the amino acid sequence of SEQ ID NO:12, LukE having the amino acid sequence of SEQ ID NO:13, or any combination thereof. 16.-20. (canceled)
 21. The binding molecule of claim 1, wherein said staphylococcal leukotoxin is selected from the group consisting of leukotoxin A (LukA), leukotoxin B (LukB), leukotoxin AB (LukAB), leukotoxin D (LukD), leukotoxin E (LukE), leukotoxin ED (LukED), Panton-Valentine leukocidin S (LukS-PV), Panton-Valentine leukocidin F (LukF-PV), Panton-Valentine leukocidin (LukSF/PVL), gamma hemolysin A (HlgA), gamma hemolysin C (HlgC), gamma hemolysin B (HlgB), gamma hemolysin AB (HlgAB), and gamma-hemolysin BC (HlgBC).
 22. (canceled)
 23. The binding molecule of claim 1, wherein the first and the second binding domains are connected via a linker peptide.
 24. The binding molecule according to claim 1, wherein the first binding domain comprises an immunoglobulin heavy chain variable (VH) region having the amino acid sequence of SEQ ID NO:60, 62, 64 or
 66. 25. The binding molecule according to claim 1, wherein the first binding domain comprises an immunoglobulin light chain variable (VL) region having the amino acid sequence of SEQ ID NO:61, 63, 65 or
 67. 26. The binding molecule of claim 1, wherein the first binding domain comprises: a VH region having the amino acid sequence of SEQ ID NO:60, 62, 64 or 66 and a VL region having the amino acid sequence of SEQ ID NO:61, 63, 65 or
 67. 27. The binding molecule according to claim 1, wherein the first binding domain comprises: (1) a VH region having the amino acid sequence of SEQ ID NO:60, and a VL region having the amino acid sequence of SEQ ID NO:61; (2) a VH region having the amino acid sequence of SEQ ID NO:62, and a VL region having the amino acid sequence of SEQ ID NO:63; (3) a VH region having the amino acid sequence of SEQ ID NO:64, and a VL region having the amino acid sequence of SEQ ID NO:65; (4) a VH region having the amino acid sequence of SEQ ID NO:66, and a VL region having the amino acid sequence of SEQ ID NO:67; (5) a VH region having the amino acid sequence of SEQ ID NO:68, and a VL region having the amino acid sequence of SEQ ID NO:69; (6) a VH region having the amino acid sequence of SEQ ID NO:70, and a VL region having the amino acid sequence of SEQ ID NO:71; (7) a VH region having the amino acid sequence of SEQ ID NO:72, and a VL region having the amino acid sequence of SEQ ID NO:73; (8) a VH region having the amino acid sequence of SEQ ID NO:74, and a VL region having the amino acid sequence of SEQ ID NO:75; (9) a VH region having the amino acid sequence of SEQ ID NO:76, and a VL region having the amino acid sequence of SEQ ID NO:77; or (10) a VH region having the amino acid sequence of SEQ ID NO:78, and a VL region having the amino acid sequence of SEQ ID NO:79.
 28. The binding molecule of claim 1, wherein the first binding domain comprises (i) a VH region having the amino acid sequence of SEQ ID NO:60, 62, 64 or 66; and (ii) a VL region having the amino acid sequence of SEQ ID NO:61, 63, 65 or 67; and the second binding domain comprises the amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666.
 29. The binding molecule of claim 1 further comprising: one or more additional binding domains, said additional binding domains capable of (i) binding to a different glycosylated staphylococcal surface protein than bound by the first binding domain and/or (ii) binding to a different staphylococcal leukotoxin than bound by the second binding domain.
 30. A nucleic acid sequence encoding the binding molecule of claim
 1. 31. A vector comprising the nucleic acid sequence of claim
 30. 32. A host cell comprising the vector of claim
 31. 33. A process for the production of a binding molecule, comprising: culturing the host cell as defined in claim 32 under conditions allowing the expression of the binding molecule, and recovering the binding molecule from the culture.
 34. A pharmaceutical composition comprising: the binding molecule according to claim 1 and a pharmaceutically acceptable carrier.
 35. (canceled)
 36. A method for the treatment, prevention, or amelioration of a staphylococcal infection, comprising: administering to a subject in need thereof the binding molecule according to claim 1 under conditions effective to treat, prevent or ameliorate the staphylococcal infection. 37.-40. (canceled)
 41. A method for diagnosing a staphylococcal infection in a subject comprising: contacting the binding molecule according to claim 1, with a sample from the subject; detecting presence or absence of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample based on said contacting; and diagnosing the staphylococcal infection in the subject based on said detecting.
 42. A method for the detection of staphylococcus in a sample, comprising: contacting the binding molecule according to claim 1 with the sample; detecting presence or absence of a glycosylated staphylococcus surface protein and/or a staphylococcal leukotoxin in the sample based on said contacting, whereby the presence of the glycosylated staphylococcus surface protein and/or staphylococcal leukotoxin indicates the presence of staphylococcus in the sample.
 43. A method for the prevention of a staphylococcal infection in a subject, comprising: contacting the binding molecule according to claim 1 with a sample from the subject; detecting a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample based on said contacting; and administering an agent suitable for preventing staphylococcal infection to the subject based on said detecting.
 44. A kit comprising a binding molecule of claim
 1. 45. A kit comprising the nucleic acid molecule of claim
 30. 